Method for producing edible crosslinked porous hollow fibers and membranes by pH-induced phase separation, and their use.

Abstract

This invention provides a method for producing edible cross-linked porous hollow fibers and sheet membranes suitable for the manufacture of clean meat products, as well as a method for using them. [Solution] The manufacturing method includes a) a step of preparing i) one or more edible proteins, ii) one or more solvents, and iii) a forming bath; the one or more solvents or the forming bath also includes one or more polyvalent cations or anions or buffers; b) a step of co-mixing the one or more edible proteins in the one or more solvents to form a mixture; c) a step of extruding the mixture into the forming bath to form extruded hollow fibers, or casting the mixture into the forming bath to form a sheet film; d) a step of exposing the extruded hollow fibers or sheet film to an energy source selected from one or more of heat and irradiation sufficient to at least partially crosslink the one or more proteins to form edible crosslinked porous hollow fibers or sheet films.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 234,796, filed Aug. 19, 2021, the entire contents of which are hereby incorporated by reference.

Background Art

[0002] The integrity and pore characteristics of a membrane are of utmost importance for its effective use in membrane - based bioreactors. The membrane needs to be self - supporting to allow for the movement of media and nutrients through the membrane without obstructing the support structure and to allow for a larger surface area for the cultivation of adherent cells. Further, for manufacturing edible foods, the membrane generally needs to be made of materials considered safe (GRAS). Even further, creating an edible membrane from both a technical aspect (i.e., non - toxic and digestible) and a practical consumer - acceptable aspect (i.e., having a texture and mouthfeel acceptable to consumers) has not been achieved in the art. Manufacturing such membranes, whether flat sheets (e.g., nanoporous membranes) or fibers (e.g., hollow fibers), has been difficult to accomplish.

Summary of the Invention

Problems to be Solved by the Invention

[0003] What is needed is a highly - integrity membrane suitable for cell culture and for use in an edible membrane - based bioreactor.

Means for Solving the Problems

[0004] The inventors have developed a novel and non-trivial method for fabricating membranes (i.e., membrane films and fibers) by pH-induced phase separation or proton-induced phase separation, possessing the structural integrity required for use in bioreactors, for example, for the production of food for human and animal consumption. These membranes are fabricated using GRAS materials, are self-supporting (i.e., do not collapse under their own weight, rip easily, or break easily when subjected to or exposed to the hydrodynamic forces required by the culture conditions in a bioreactor), and are edible in both technical and practical terms, making them acceptable to consumers.

[0005] In its broadest embodiment, the membrane of the present invention comprises one or more plant or animal proteins, one or more edible polysaccharides, and optionally one or more polysaccharide crosslinking agents. The proteins, polysaccharides, and optionally selected crosslinking agents are co-mixed and extruded into a forming bath. The forming bath contains one or more ions (i.e., cations or anions) that result in crosslinking of polysaccharides in the membrane. Furthermore, in some embodiments of the present invention, a change in pH in the forming bath results in phase separation-inducing membrane formation.

[0006] The inventors have empirically learned that crosslinking of polysaccharides in membranes is often insufficient to ensure adequate membrane integrity, particularly under cell culture conditions (see example). The inventors have further invented a method for imparting the necessary integrity to a membrane. After forming the membrane in a forming bath, the membrane is exposed to an energy source such as heat or irradiation. While not limited by theory, the inventors believe that exposure to an energy source results in crosslinking of polysaccharides and / or proteins in the membrane, thereby providing the necessary integrity to the membrane while maintaining the quality required for consumer acceptance.

[0007] Furthermore, with regard to providing membranes for use in food, prior art involving chemical crosslinking often uses toxic compounds that need to be avoided for this application. Alternatively, prior art polymer modification techniques may be used to increase the number of crosslinking sites, but may face regulatory challenges.

[0008] In another embodiment, the film of the present invention can be coated with one or more agents or otherwise modified to enhance, for example, cell adhesion and cell proliferation. The film can be coated before or after exposure to heat or irradiation.

[0009] After formation, exposure to an energy source, and optional coating, the film may be partially dried and / or stored, or subjected to further processing (e.g., by cutting to size and incorporating into a bioreactor cartridge or capsule).

[0010] Accordingly, the present invention relates to edible 3D nano- and microporous structures for use in membrane bioreactors (film or fiber-based) for producing, for example, structured clean meat products. Culture media pass through the membrane, and cells are grown on one or both sides of the membrane. Prior art hollow fiber membrane bioreactors exist for adherent cells, but require trypsin or other chemical / enzymatic processes to remove the cells. Such processes are too expensive for commercial-scale clean meat production and, furthermore, destroy any tissue-like structures. Accordingly, the present invention envisions a membrane to be consumed together with meat cells used in the production of cultured meat products. The present invention further envisions a membrane that is at least partially soluble. This aspect may be required, for example, to achieve a desired texture in the final structured meat product.

[0011] Food-based materials for adherent cell scaffolds have been described in the art. However, these material formats are not suitable for (hollow fiber) membrane bioreactors. These material formats are generally non-porous films, fiber-based mats (such as electrospinning or rotary jet spinning), or sponges (usually derived from freeze-drying, extrusion, and / or foaming processes).

[0012] Membrane bioreactors require highly specific pore sizes with particular membrane geometric shapes. Hollow fiber bioreactors (HFBRs) typically have pore sizes between 5 kDa and 0.1 μm, depending on the cell type, bioreactor design, and bioprocess.

[0013] Although this invention intends for hollow fibers, the general concepts of this invention can also be applied to flat sheet (film-like) membranes. Sheet membranes are formed, for example, by casting a polymer onto a sacrificial surface and then immersing this sacrificial surface in a bath designed to solidify the polymer. Hollow fibers are formed by spinning them into the bath from a nozzle / spinneret. When manufacturing hollow fibers, as is known to those skilled in the art, the bore fluid must also be precisely determined and controlled. Further details regarding the manufacture of sheet membranes and hollow fibers are given below.

[0014] The method invented by the present inventors for producing the membrane of the present invention utilizes multiple steps. A high protein content is preferred for human nutritional considerations and cell adhesion considerations. However, the molecular weight of the protein is generally too low to provide sufficient chain entanglement or structural integrity for fiber formation properties. For this reason, an additional “carrier” polymer is added to the membrane polymer (i.e., a doped solution). As taught herein, the carrier polymer is a polysaccharide selected from, for example, one or more of alginates, cellulose, pectin, chitin, chitosan, gellan gum, xanthan gum, arabinoxylan, glucomannan and others known to those skilled in the art.

[0015] Proteins and polysaccharides are mixed in a blend of GRAS solvents. One or more proteins and one or more polysaccharides are selected, and once the mixture is formed, they are solidified in a solidification bath to instantly or nearly instantly fix the dimensions of the film to be cast. In one embodiment, the bath is intended to contain polyvalent cations such as Ca2+ and Mg2+. Specifically, the inventors have demonstrated that Ca2+ instantaneously crosslinks alginates, pectins, or other polysaccharides in the film. This fixes the dimensions of the fiber / sheet, achieving the desired three-dimensional target.

[0016] However, at this point, the proteins are not crosslinked, only the polysaccharides are ionically crosslinked. As described in the literature and observed in practice, ionically crosslinked polysaccharides can dissociate in cell culture media. Therefore, when used in cell culture, an additional crosslinking step is required to further enhance membrane stability and ensure membrane integrity. Since covalent crosslinking requires harsh chemicals, this method is undesirable for food products. The innovation of the present invention is the use of physical crosslinking, which is generated via an energy source such as heat, gamma, electron beam, beta, X-ray, or UV (one or more of these). Since these are used in the food industry to kill or weaken potential pathogens, it will be understood by those skilled in the art that they are safe for use in food.

[0017] An alternative method is further intended by the present invention to be the use of protein crosslinking agents already approved for food applications, such as transglutaminase. In addition to, or instead of, protein crosslinking, it is also intended that the potential crosslinking sites on the polymer can be increased by modifying polysaccharides before creating the mixture.

[0018] The present invention further intends to explore other methods, such as directly dissolving proteins in an alcohol / water blend and solidifying the membrane in an acid bath. The present invention even further intends to dissolve plant protein isolates in an alkaline solution and then solidify them with an organic coagulant, such as alcohol or a neutralizing acid / caustic solution. For example, when chitosan is dissolved in 5% acetic acid and extruded into a bath with a higher pH, the polymer solidifies into a fibrous shape.

[0019] Chitosan can also be dissolved in a slightly acidic bath (such as about 5% acetic acid or citric acid), and then deposited / spun into a bath containing a certain concentration of tripolyphosphate / sodium tripolyphosphate (TPP) that maintains and / or preserves the porosity of the solidified chitosan. The bore fluid may also contain a solution similar to the bath solution.

[0020] Chemical or enzymatic crosslinking agents can also be added to the bore fluid (the fluid used in a nozzle bore when forming solid or hollow fibers; bore fluids are known to those skilled in the art) and / or the forming bath to help crosslink plant proteins in polysaccharide and protein blends. Examples of crosslinking agents that may be optionally included in the bath or bore fluid are transglutaminase, tripoliphosphate, genipin (genipin is a chemical compound found in the fruit extract of Genipa americana), or other oxidases known to those skilled in the art.

[0021] Another aspect of the present invention is that fibers can be impregnated insoluble (at least in the solvent system used) into a doping solution (i.e., a mixture of proteins and polysaccharides). These fibers may be, for example, bacterial nanocellulose, nanocellulose, or other suitable fibers. These fibers can perform two functions: the first is mechanical reinforcement resulting in an increase in "toughness" as defined by a stress-strain curve chart; the second function of these fibers would be to promote myotubular alignment; during extrusion, these fibers naturally align with the hollow fibers, and the fibers on the surface of the hollow fiber membrane promote the alignment of cells that have grown there.

[0022] Another aspect of the present invention is the geometry and topography of the fiber itself. Preferably, the fiber has an outer diameter of from about 300 to about 700 microns. Striations or grooves extending parallel, substantially parallel or essentially parallel to the fiber length are desired features and structural features that can be incorporated into fibers made by the methods of the present invention. Striations or grooves along the fiber can be incorporated into the spinning method through the formulation and mixing of the dope solution, through the geometry of the nozzle, or through turbulence in the forming bath by methods known to those skilled in the art.

[0023] It is further contemplated that another step of this method can be to increase cell adhesion on the membranes and fibers by using the desired chemical processes or compounds that change the surface of the membranes or fibers or coat the membranes or fibers. Examples of suitable processes and compounds include, but are not limited to, plasma treatment, proteins including, but not limited to, fibronectin, fibrinogen, laminin, collagen, gelatin, etc., or addition of cell binding sites through addition of short-chain peptide sequences isolated from those proteins including, but not limited to, RGD, YIGSR, IKVAV, DGEA, PHRSN, PRARI, etc.

[0024] Coatings are contemplated that can also be applied to target uses beyond cell adhesion. Heparin can increase the concentration of growth factors on the fiber surface. Compounds that assist in cell differentiation can also be applied. For example, coatings having a high lipid content can promote the differentiation of appropriate cells into adipocytes.

[0025] Coatings directed to non-biological (i.e., not directly related to the growth and maintenance of the desired cells) results are also contemplated. Preservatives and / or antibiotics can be used to prevent spoilage or maintain a sterile environment before and during culturing. Dyes, pigments, beta-carotene, etc. can be applied as coatings or directly to the fiber dope solution to obtain the desired appearance. Similarly, flavors and fragrances can be applied as coatings or directly to the fiber dope solution to obtain the desired flavor profile. Plasticizers (e.g., sugar alcohols such as sorbitol and glycerol) can be applied as coatings or directly to the dope solution or bore fluid. Plasticizers enhance handling, minimize pore collapse, extend shelf life, and change the mouthfeel.

[0026] The present invention also includes membranes (hollow fibers and sheet membranes) made by the method of the present invention.

[0027] The present invention is a method for producing edible crosslinked porous hollow fibers and membrane sheets, comprising: a) preparing i) one or more edible proteins, ii) one or more edible polysaccharides, iii) one or more solvents, and iv) a forming bath, wherein one or more of the solvents or the forming bath also contains one or more polyvalent cations or anions; b) co-mixing one or more edible proteins and one or more edible polysaccharides in one or more solvents to form a mixture; c) extruding the mixture into the forming bath to form extruded hollow fibers or casting the mixture onto the bath to form a membrane sheet; and d) exposing the extruded hollow fibers or membrane sheet to an energy source selected from one or more of heat and irradiation sufficient to at least partially crosslink one or more proteins to form edible crosslinked porous hollow fibers.

[0028] The method further contemplates that one or more proteins are selected from the group consisting of pea, soybean, wheat, squash, rice, brown rice, sunflower, canola, chickpea, lentil, lupin, fava bean, corn, oat, potato, quinoa, sorghum, and peanut.

[0029] This method further aims to select one or more polysaccharides from the group consisting of agar, chitosan, chitin, alginate, sodium alginate, cellulose, hydroxypropylcellulose, methylcellulose, hydroxypropylmethylcellulose, gellan gum, xanthan gum, pectin, tapioca, guar gum, and bean gum.

[0030] This method further intends that one or more solvents be selected from the group consisting of water, acetic acid, citric acid, lactic acid, phosphoric acid, malic acid, tartaric acid, sodium hydroxide, ethanol, glycerin, and propylene glycol.

[0031] This method further aims to ensure that the ions are selected from the group consisting of Ca2+, Mg2+, Fe3+, Zn2+, tripolyphosphate, and trisodium citrate, and that the selected ions can at least enable partial crosslinking of one or more polysaccharides.

[0032] This method involves applying heat of approximately 120°C to 140°C, under a pressure of approximately 0 PSI to 20 PSI gauge, and at a relative humidity of approximately 50% to 100% for approximately 2 to 60 minutes, or further involving immersion of the fibers in a water bath of approximately 60°C to 100°C under atmospheric conditions.

[0033] This method further intends that the irradiation is selected from the group consisting of electron beam, UV light, and gamma ray irradiation, that the irradiation is applied during or after the process, and that the irradiation is about 1 to about 100 kGy or about 10 to about 50 kGy.

[0034] This method further assumes that the porosity of the hollow fiber or membrane sheet is approximately 1% to approximately 90% or approximately 50% to approximately 80%.

[0035] This method further intends to include a step of enhancing cell adhesion by coating edible cross-linked porous hollow fibers with a coating.

[0036] This method further intends that the coating be selected from one or more short-chain peptide sequences isolated from fibronectin, fibrinogen, laminin, collagen, gelatin, or these proteins.

[0037] This method further aims to select the short-chain peptide sequence from the group consisting of RGD, YIGSR, IKVAV, DGEA, PHRSN, and PRARI.

[0038] The present method further comprises a step of modifying the outer surface of edible cross-linked porous hollow fibers to enhance cell adhesion, wherein the surface modification is selected from one or more of the following: plasma, corona, abrasion, etching, ablation, or sputter coating.

[0039] This method further aims to ensure that the protein is powdered or finely ground before it dissolves in the solvent.

[0040] This method further aims to ensure that the protein is at least 70%, 80%, 90%, 95%, 98%, 99%, and 99.9% pure.

[0041] This method further aims to ensure that the polysaccharides are at least 70%, 80%, 90%, 95%, 98%, 99%, and 99.9% pure.

[0042] This method further intends for the ratio of protein to polysaccharide in the mixture to be approximately 10:1 to approximately 1:10, or approximately 4:1 to approximately 1:4. This method further intends for the ratio of protein to polysaccharide in the mixture to be approximately 1:1. This method further intends for the ratio of protein to polysaccharide in the mixture to be approximately 1:7 or approximately 7:1. In some cases, the solid ratio between protein and polysaccharide is 100:1 or approximately 1:100, or exclusively 100% protein isolate.

[0043] This method further intends that the forming bath contains RO (reverse osmosis) water in which calcium chloride is dissolved at a concentration of, for example, 15 g / L or approximately 15 g / L, but the desired concentration may be approximately 4 g / L to approximately 20 g / L, approximately 12 g / L to approximately 18 g / L, or approximately 14 g / L to approximately 16 g / L. In the continuous process, the forming bath has a supply and bleed system, and the prepared 15 g / L calcium chloride is supplied to the side of the bath and the bath is bled at the same rate.

[0044] This method further intends that the forming bath contains RO water containing one or more of the following: i) water, acetic acid, citric acid, lactic acid, phosphoric acid, malic acid, and tartaric acid, or ii) sodium hydroxide and potassium hydroxide, in combination with one or more of the following: calcium, zinc, magnesium, iron, and potassium.

[0045] This method is intended to produce edible crosslinked porous hollow fibers and membrane sheets, comprising the steps of: a) preparing i) one or more edible proteins, ii) one or more edible polysaccharides, iii) one or more solvents, and iv) a forming bath, wherein the forming bath is mainly water and further comprises 1) one or more of acetic acid, citric acid, lactic acid, phosphoric acid, malic acid, tartaric acid, or other suitable acids, or 2) one or more of sodium hydroxide and potassium hydroxide, or other suitable bases, to further contain one or more of calcium chloride, zinc chloride, magnesium ions, and potassium; b) co-mixing one or more edible proteins and one or more edible polysaccharides in one or more solvents to form a mixture; c) extruding the mixture into the forming bath to form extruded hollow fibers, or casting the mixture onto the bath to form a membrane sheet; and d) exposing the extruded hollow fibers or membrane sheet to an energy source selected from one or more of heat and irradiation sufficient to at least partially crosslink one or more proteins to form edible crosslinked porous hollow fibers. In this embodiment, ions are replenished in the forming bath.

[0046] This method further relates to any hollow fiber or sheet membrane (i.e., membrane sheet) produced by the method of the present invention.

[0047] The present invention further relates to membranes or clean meat, structured meat, cultured meat, laboratory cultured meat, cultivated meat, cell-based meat, etc., produced using the present invention, and methods for producing these meats.

[0048] The present invention is intended to be a method for producing edible crosslinked porous hollow fibers or sheet membranes, comprising the steps of: a) providing i) one or more edible proteins, ii) one or more solvents, and iii) a forming bath; wherein one or more solvents or forming baths also include one or more polyvalent cations or anions or buffers; b) co-mixing one or more edible proteins in one or more solvents to form a mixture; c) extruding the mixture into a forming bath to form extruded hollow fibers, or casting the mixture into a forming bath to form a sheet membrane; and d) exposing the extruded hollow fibers or sheet membranes to an energy source selected from one or more of heat and irradiation sufficient to at least partially crosslink one or more proteins to form edible crosslinked porous hollow fibers or sheet membranes.

[0049] The present invention is further intended to involve preparing one or more edible polysaccharides and co-mixing one or more polysaccharides with one or more edible proteins in one or more solvents.

[0050] The present invention is further intended to relate to the preparation of a plasticizer and the co-mixing of the plasticizer with one or more edible proteins in one or more solvents.

[0051] It is further intended that the method of the present invention relates to selecting one or more proteins from the group consisting of peas, soybeans, wheat, pumpkins, rice, brown rice, sunflowers, canola, chickpeas, lentils, mung beans, white beans, corn, oats, potatoes, quinoa, sorghum, and peanuts.

[0052] It is further intended that the method of the present invention relates to the selection of one or more polysaccharides from the group consisting of agar, chitosan, chitin, alginate, sodium alginate, cellulose, hydroxypropylcellulose, methylcellulose, hydroxypropylmethylcellulose, gellan gum, xanthan gum, pectin, tapioca, guar gum, and bean gum.

[0053] It is further intended that the method of the present invention relates to one or more solvents being selected from the group consisting of water, acetic acid, citric acid, lactic acid, phosphoric acid, malic acid, tartaric acid, sodium hydroxide, ethanol, glycerin, and propylene glycol.

[0054] It is further intended that the method of the present invention relates to a forming bath containing one or more of the following in combination with 1) water, acetic acid, citric acid, lactic acid, phosphoric acid, malic acid, and tartaric acid, or 2) sodium hydroxide and potassium hydroxide, respectively, one or more of the following: calcium, zinc, magnesium, iron, and potassium.

[0055] It is further intended that the method of the present invention relates to the selection of the ion from the group consisting of Ca2+, Mg2+, Fe3+, Zn2+, tripolyphosphate, and trisodium citrate, wherein the selected ion can at least enable partial crosslinking of one or more polysaccharides.

[0056] The present invention is further intended to relate to the heating of the mixture in step b).

[0057] It is further intended that the method of the present invention relates to heating the formed hollow fiber or sheet membrane at approximately 70°C to approximately 140°C or approximately 120°C to 140°C for approximately 2 to approximately 60 minutes under a pressure of approximately 0 PSI to approximately 20 PSI gauge and a relative humidity of approximately 50% to approximately 100%, or to immersing the hollow fiber or sheet membrane in a water bath at approximately 60°C to approximately 100°C under atmospheric conditions.

[0058] It is further intended that the method of the present invention relates to the co-mixing being carried out at a temperature of approximately 0°C to approximately 90°C.

[0059] It is further intended that the method of the present invention relates to the fact that the mixture has a pH of about 10 to about 13 and the compounded bath has a pH of about 3 to about 5.

[0060] The present invention further intends to be related to the neutralization of the film to a pH of approximately 6.8 to 7.8 after formation.

[0061] The present invention further intends to relate to the neutralization of the film to a pH of approximately 7.3 to 7.5 after its formation.

[0062] It is further intended that the method of the present invention relates to the selection of irradiation from the group consisting of electron beam, UV light, and gamma ray irradiation.

[0063] The present invention is further intended to relate to a method in which irradiation is applied during or after the process. The present invention is further intended to relate to a method in which irradiation is about 1 to about 100 kGy or about 10 to about 50 kGy.

[0064] It is further intended that the method of the present invention relates to a hollow fiber or sheet membrane having a porosity of about 1% to about 90%, about 25% to about 75%, or about 40% to about 60%.

[0065] It is further intended that the method of the present invention relates to a hollow fiber or sheet membrane having a porosity of about 50% to about 80%.

[0066] This method is further intended to include a step of enhancing cell adhesion by coating edible cross-linked porous hollow fibers or sheet membranes with a coating.

[0067] The present invention is further intended to relate to a method in which the coating is selected from one or more short-chain peptide sequences isolated from fibronectin, fibrinogen, laminin, collagen, gelatin, or these proteins.

[0068] It is further intended that the method of the present invention relates to one or more short-chain peptide sequences selected from the group consisting of RGD, YIGSR, IKVAV, DGEA, PHRSN, and PRARI.

[0069] The present invention is further intended to relate to a method for modifying the outer surface of edible cross-linked porous hollow fibers to enhance cell adhesion. The present invention is further intended to relate to a method further comprising the step of coating edible cross-linked porous hollow fibers or sheet films with a plasticizer. The present invention is further intended to relate to a surface modification being selected from one or more of plasma, corona, abrasion, etching, ablation, or sputter coating.

[0070] The present invention is further intended to relate to a method in which the protein is powdered or pulverized before being dissolved in a solvent.

[0071] It is further intended that the method of the present invention relates to the protein being at least 70%, 80%, 90%, 95%, 98%, 99%, or 99.9% pure.

[0072] It is further intended that the method of the present invention relates to the polysaccharide being at least 70%, 80%, 90%, 95%, 98%, 99%, or 99.9% pure.

[0073] The present invention is further intended to relate to a method in which the ratio of protein to polysaccharide (protein:polysaccharide) in the mixture is approximately 10:1 to approximately 1:10 or approximately 1:99 to approximately 99:1, 98:2, 97:3, 96:4, 95:5 or 90:10. The present invention is further intended to relate to a ratio of protein to polysaccharide in the mixture is approximately 4:1 to 1:4. The present invention is further intended to relate to a ratio of protein to polysaccharide in the mixture is approximately 1:1 or 7:1.

[0074] The present invention is further intended to relate to a forming bath containing one or more of the following in combination with i) water, acetic acid, citric acid, lactic acid, phosphoric acid, malic acid, and tartaric acid, or ii) sodium hydroxide and potassium hydroxide.

[0075] It is further intended that the present invention relates to hollow fibers or sheet membranes produced by any of the methods of the present invention.

[0076] The present invention is intended to be a method for producing edible crosslinked porous hollow fibers or sheet membranes, comprising the steps of: a) providing i) one or more edible proteins, ii) one or more edible polysaccharides, iii) one or more solvents, and iv) a forming bath, wherein the forming bath contains 1) one or more of water, acetic acid, citric acid, lactic acid, phosphoric acid, malic acid, tartaric acid, or 2) one or more of sodium hydroxide and potassium hydroxide, in combination with one or more of calcium, zinc, magnesium, iron, and potassium; b) co-mixing one or more edible proteins and one or more edible polysaccharides in one or more solvents to form a mixture; c) extruding the mixture into the forming bath to form extruded hollow fibers, or casting the mixture to form a sheet membrane; and d) exposing the extruded hollow fibers or sheet membrane to an energy source selected from one or more of heat and irradiation sufficient to at least partially crosslink one or more proteins to form edible crosslinked porous hollow fibers.

[0077] The present invention is intended to be a method for producing hollow fibers or sheet membranes in which one or more proteins, one or more polysaccharides, one or more solvents, plasticizers and / or one or more components of a forming bath are generally recognized as safe (GRAS) by the U.S. Food and Drug Administration (FDA).

[0078] It is further intended that the present invention relates to a film or hollow fiber obtained by any of the methods of the present invention undergoing an exchange of 10-50% water for glycerol for drying, wherein the drying does not result in pore breakdown. [Brief explanation of the drawing]

[0079] [Figure 1] This is a schematic diagram of a method used to produce the membrane and hollow fibers of the present invention. [Figure 2] This is a schematic diagram of another method used to produce the membrane and hollow fibers of the present invention. [Figure 3A] This figure shows a hollow fiber membrane manufactured by the method of the present invention. [Figure 3B] This figure shows a hollow fiber membrane manufactured by the method of the present invention. [Figure 4A] This figure shows a scanning electron microscope (SEM) image of fibers produced by the method of the present invention. [Figure 4B] This figure shows a scanning electron microscope (SEM) image of fibers produced by the method of the present invention. [Figure 4C] This figure shows scanning electron microscope (SEM) images of fibers produced by the method of the present invention. A shows that the surface pores of the whey protein and alginate blend are approximately 20 nm or about 1000 kDa. This image also shows that the fringes from the method are parallel to the length of the fibers. B shows the surface pores of the pumpkin protein isolate and alginate blend having surface pores of approximately 100 nm or less. C shows a low-resolution image of fibers made using the pumpkin protein isolate. [Figure 5A] This figure shows fibers produced by the method of the present invention. [Figure 5B] This figure shows fibers produced by the method of the present invention. The hollow fibers of the present invention can easily support the weight required in a bioreactor. (A) The shown fiber is 2 meters long. (B) The fibers produced by the method of the present invention can support at least 9 grams. [Figure 6] This figure shows mung bean cast film from urea and sodium hydroxide solutions. The image shows the mung bean doped solution cast onto glass via doctor blade technology. It can be seen that the doped solution is transparent before solidification. [Figure 7] This figure shows viscosity measurements taken using a Brookfield (Middleboro, Massachusetts) viscometer equipped with an S64 spindle. Viscosities of 2% alginate and 10% protein isolates are shown. Each mixture had a pH adjusted to pH 11 before measurement. [Figure 8] This is a diagram showing a simple design plot in terms of quantity. This is an experimental design using Minitab (Pennsylvania State University) to observe water containing urea, ethanol, and sodium hydroxide. [Figure 9] Figure 8 shows the temperature sweep of 15% zein in the solvent blend. This indicates that a solvent system containing a low concentration of ethanol, around 12.5%, can dissolve zein. [Figure 10] This figure shows that by using the solvent conditions shown in Figure 8, the gelation properties of agarose can be changed compared to the same agarose in water. [Figure 11] This figure shows that zein and agarose can be blended using the given solvent system shown in Figure 8, within a given mixing temperature range, particularly above 40°C, without solidifying any of the components. [Figure 12A] This diagram shows an image of the zein film manufacturing method, which consists of a film casting process (left). [Figure 12B] This image shows a zein membrane manufacturing method consisting of a coagulation step (right) in acetate buffer (0.2M, pH 4.5). [Figure 13]This figure shows the crosslinking process of a mung beet alginate film using a hot glycerol bath set to 120°C for one hour. [Figure 14A] This figure shows a graph illustrating the elastic modulus of the membrane (left). [Figure 14B] This figure shows a graph illustrating the strain on the membrane (right). [Figure 15A] This diagram shows the elastic moduli of various tissues (left). [Figure 15B] This figure shows the elastic modulus of an exemplary membrane material of the present invention (right). [Figure 16] This figure shows images (1-6) of membranes manufactured according to different manufacturing protocols in order to investigate and verify each manufacturing process. AC represents a "0.2 M acetate bath at pH 4.5", H represents a "HEPES buffer" (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) bath at pH 7.4, G represents a "glycerol bath", HG represents a "hot glycerol bath", HW represents "hot water" (autoclave), and 0 represents "no process performed". [Figure 17] Figures 17A and 17B show the elastic modulus of the film (A: left) and the fracture strain of the film (B: right), respectively. Sample 6 was manufactured according to protocol AC-0-G-HG, sample 5 according to protocol AC-0-0-HG, and sample 2 according to protocol AC-0-0-HW. [Figure 18] Figures 18A-C show the changes in elastic modulus (A: left), strain (B: center), and final stress (C: right) of heat-treated mung bean films (glycerol-based protocol) as the solidification time in an acetic acid bath increases. The figures show the mechanical properties of films solidified for 10 minutes to a maximum of 3 hours. [Figure 19] Figures 19A-C show the changes in elastic modulus (A: left), strain (B: center), and final stress (C: right) of mung bean film as the heat treatment time based on glycerol increases. The heat treatment based on glycerol was investigated by keeping the duration of both the solidification bath (10 minutes) and the water-glycerol exchange (10 minutes) constant, and varying the duration of the heat treatment after reaching the final temperature of 120°C. [Figure 20]This figure shows a rheological investigation of the heat treatment of mung bean films using glycerol. The graph shows the change in tangent delta (δ) with respect to the temperature gradient. [Figure 21] Figures 21A-C show the changes in (A) modulus of elasticity, (B) strain, and (C) final stress as the heat treatment time of mung bean film based on glycerol increases. [Figure 22] This figure shows the elastic modulus values ​​of an alginate and gluten protein blend containing wheat gluten, mung bean, and zein when incubated in cell medium at 37°C. Mechanical tensile measurements were performed before and after incubation for 3, 10, and 21 days. [Figure 23] This figure shows the breaking strain values ​​of an alginate and gluten protein blend containing wheat gluten, mung bean, and zein when incubated in cell medium at 37°C. Mechanical tensile measurements were performed before and after incubation for 3, 10, and 21 days. [Figure 24] This figure shows the membrane surface area of ​​an alginate protein blend containing wheat gluten, mung bean, and zein when incubated in cell medium at 37°C. Measurements were taken before and after incubation for 3, 10, and 21 days. [Figure 25] This figure shows the membrane surface area of ​​an agarose protein blend containing wheat gluten, mung bean, and zein when incubated in cell medium at 37°C. Measurements were taken before and after incubation periods of 3, 10, and 21 days. [Figure 26] Figures 26A and 26B show a comparison of (A) modulus of elasticity and (B) strain between brown rice-alginate blends prepared with and without transglutaminase crosslinking, before and after incubation for 3, 10, and 21 days in cell culture medium at 37°C. [Figure 27]Figures 27A-F show the elastic modulus (A and B: left), fracture strain (B and E: center), and surface area (C and F: right) of protein membranes containing soy protein isolates (A-C: top) and mung bean (D-F: bottom). Measurements were taken before and after incubation of soy protein isolates in cell medium at 37°C for 3, 10, and 21 days, and before and after incubation of mung bean in cell medium at 37°C for 5, 12, and 30 days. [Figure 28] This figure shows scanning electron microscope images of the membrane surface (top) and cross-section (bottom) of a soy protein isolate. [Figure 29] This figure shows scanning electron microscope images of the membrane surface (top) and cross-section (bottom) of a mung beetle protein isolate. [Figure 30] This figure shows scanning electron microscope images of the membrane surface (top) and cross-section (bottom) of a zein protein isolate, as well as the membrane surface (top) and cross-section (bottom) of a zein protein isolate and agarose membrane. [Figure 31] This figure shows scanning electron microscope images of the surface and cross-section of zein alginate (left) and pea protein-k-carrageenan (right) membranes. [Figure 32] This figure shows scanning electron microscope images of the surface and cross-section of mung bean-agarose (left) and soybean-alginate (right) membranes. [Figure 33] This figure shows scanning electron microscopy images of the cross-section (top) and surface (bottom) of mung bean-alginate hollow fiber. [Figure 34] This figure shows fluorescent cell adhesion and proliferation tests performed using the C2C12 cell line on zein, soybean, and mung bean TG-crosslinked mung bean membranes. A live (green) / dead (red) assay was performed after a 48-hour growth period. The micrograph shows almost no red staining, indicating that nearly all cells are viable. [Figure 35]This figure shows cell fluorescence adhesion and proliferation tests performed on mung bean and chitosan-coated membranes using the C2C12 cell line. A live (green) / dead (red) assay was performed after a 48-hour growth period. The micrograph shows almost no red staining, indicating that nearly all cells are viable. [Figure 36] This figure shows fluorescence cell adhesion and proliferation tests performed on heat-treated and unheat-treated soybean-alginate, peanut-alginate, and zein-agarose membranes using the C2C12 cell line. A live (green) / dead (red) assay was performed after a 48-hour growth period. The micrograph shows almost no red staining, indicating that nearly all cells are viable. [Figure 37] This figure shows a fluorescence cell adhesion and proliferation test performed using the QM7 cell line on soybean, mung bean coated with fibronectin and collagen, and chitosan membranes. A live (green) / dead (red) assay was performed after a 48-hour growth period. The micrograph shows almost no red staining, indicating that nearly all cells are viable. [Figure 38] This figure shows the effects of drying and rehydration on alginate:mung bean-based films. [Modes for carrying out the invention]

[0080] structured meat products The present invention, however, envisions, for example, an edible membrane containing a solid hollow fiber suitable for use in a bioreactor to produce structured clean meat, a method for producing structured clean meat using the same, and structured clean meat produced using the hollow fiber of the present invention. Clean meat (also known in the art as “cultured meat” or “lab-cultured meat”) is defined in the art as meat or meat-like products grown from cells in a laboratory, factory, or other manufacturing facility suitable for large-scale cell culture (collectively referred to herein as “clean meat” or “clean meat products”).

[0081] "Structured meat products," "structured clean meat products," "structured cultured meat," or "structured cultured meat products" are meat products or clean meat products that have a texture and structure similar to or reminiscent of natural animal-derived meat, such as natural animal-derived meat. The structured meat products of the present invention have a texture and structure similar to natural meat in 1) texture and appearance, 2) handling when prepared for cooking and consumption (e.g., when sliced, ground, cooked, etc.), and 3) mouthfeel when consumed by humans. When the materials and methods of the present invention are used in the production of structured clean meat, they achieve at least one of these criteria, two of these criteria, or all three of these criteria. The prior art cannot produce structured meat products that adequately meet any of these criteria.

[0082] The structured meat product of the present invention meets these criteria by culturing appropriate cells (as described below) in a bioreactor (as described below) containing the hollow fibers of the present invention. The hollow fibers of the present invention provide the final structured clean meat product with a structure and texture that provides the desired appearance, handling and mouthfeel of the product, at least in a substantial part. Furthermore, the hollow fibers of the present invention help to provide a suitable environment for cell growth into the structured clean meat product. In this regard, the hollow fibers of the present invention provide a surface suitable for at least the adhesion of cultured cells, the elongation of cells into a form similar to myocytes or myocyte-like cells (i.e., substantially similar to myocytes in structure and appearance), and the formation of myocytes into sarcotubules or sarcotubule-like structures (i.e., substantially similar to sarcotubules in structure and appearance).

[0083] Manufacturing of the film of the present invention In the present invention, the terms “membrane” or “membranes” are understood to refer to any porous membrane structure produced by the methods of the present invention, including, but not limited to, hollow fiber membranes and sheet (i.e., flat) membranes. Unless otherwise specifically indicated, references to “membrane,” “hollow fiber,” “hollow fiber membrane,” and “sheet membrane” are understood to include any membrane structure produced by the methods of the present invention, regardless of shape, form, or appearance.

[0084] An exemplary manufacturing method is shown in a schematic form in Figures 1 and 2.

[0085] The edible and / or soluble hollow fibers and sheet membranes of the present invention are intended to be made from one or more of the following: hydrophilic colloids (i.e., polysaccharides such as xanthan gum, methylcellulose, alginate, agar, pectin, gelatin, carrageenan, cellulose / gellan / guar / cod / bean / other gums), proteins (e.g., polypeptides, peptides, glycoproteins and amino acids; e.g., various starches (corn / potato / rice / wheat / sorghum), plant isolates (e.g., soybean / zein / casein / wheat / mung bean protein), lipids (e.g., free fatty acids, triglycerides, natural waxes and phospholipids), alcohols (e.g., polyalcohols), carbohydrates and other natural substances, e.g., alginate). Furthermore, other materials that aid in cell adhesion and cell proliferation may be added to the hollow fibers. It is intended that the materials be added to or coated onto the hollow fibers. For example, the hollow fiber additive or coating is intended to be one or more of the following: proteins, hydrogels, or other coatings known to those skilled in the art, including extracellular matrix (ECM) components and extracts isolated from plants or synthesized from simpler substances, poly-D-lysine, laminin, collagen (e.g., collagen I and collagen IV), gelatin, fibronectin, plant-based ECM materials, collagen-like, fibronectin-like, and laminin-like materials. The overall result is that the fibers of the present invention impart the texture and structure of meat and meat products, giving the structured clean meat products produced by the present invention a texture, appearance, handling, and mouthfeel similar to actual meat.

[0086] The inventors of the present invention have acknowledged that soybean and mung bean protein isolates impart some of the desired properties to membranes produced by the method of the present invention. The inventors have also acknowledged that both soybean (Glycine max) and mung bean (Vigna radiata) belong to the same taxonomic family, Fabaceae, which is related to leguminous plants (i.e., peas or beans). Doyle, JJ, Leguminosae, Encyclopedia of Genetics, 2001, 1081-1085. While the present invention is not theoretically limited, other members of this family, particularly millettioids and phaseoloids including the genera Glycine and Vigna, are thought to act substantially similarly to soybean and mung bean protein isolates. See Figure 39.

[0087] More specifically, the hollow fibers of the present invention may contain one or more of the following: cellulose, chitosan, collagen, zein, alginate, agar, inulin, gluten, pectin, leguminous plant proteins, methylcellulose, gelatin, tapioca, xanthan gum / guar / tara / bean / other gums, proteins (e.g., but not limited to various forms of corn / potato / rice / wheat / sorghum starch, plant isolates and polypeptides, peptides, glycoproteins and amino acids including soybean / zein / casein / wheat proteins (all of which are known to those skilled in the art)), and lipids (e.g., free fatty acids, triglycerides, natural waxes and phospholipids). Cellulose polymers may include cellulose acetate butyrate, cellulose propionate, ethylcellulose, methylcellulose, nitrocellulose, etc. More specifically, the hollow fibers of the present invention may contain a mixture of one or more leguminous plant proteins and hydrophilic colloids.

[0088] In some embodiments, the hollow fibers of the present invention are intended to be edible, soluble, or edible and soluble. In other words, the fibers may be edible, soluble, or both. Furthermore, for soluble fibers, various degrees of solubility may exist. For example, some fibers may be readily soluble upon exposure to a suitable solvent (e.g., a non-toxic solvent generally considered safe by the U.S. Food and Drug Administration (FDA) or other organizations recognized as qualified to assess the safety of consumables). Other fibers may not be very readily soluble. In this regard, after the cultured cells have reached the required level of culture density, less readily soluble fibers may be partially dissolved, thereby leaving enough fiber to provide the desired mouthfeel and texture to the structured clean meat of the present invention, but without leaving excessive fiber that might make the structured clean meat product of the present invention appear chewy or difficult to chew. Soluble hollow fiber components are known to those skilled in the art. For example, alginates are soluble upon exposure to Ca2+ chelating agents. In one embodiment of the present invention, the hollow fibers of the present invention are intended to contain an amount of alginate that makes the fibers partially soluble, and / or a certain percentage of the fibers in an apparatus containing the hollow fibers of the present invention contain alginate.

[0089] In some embodiments of the present invention, one or more crosslinking agents are intended to be used in the hollow fibers of the present invention. As the name suggests, the crosslinking agents strengthen the fibers by binding one or more of the other components of the hollow fibers. In some embodiments of the present invention, the crosslinking agent may be a soluble component of the hollow fibers of the present invention or one of the soluble components. Exemplary crosslinking agents and crosslinking mechanisms intended by the present invention include, but are not limited to, covalent ester crosslinking (U.S. Patent No. 7,247,191) and UV crosslinking (U.S. Patent No. 8,337,598), both of which are incorporated herein by whole reference. Furthermore, the use of crosslinking agents in the manufacture of hollow fibers is known to those skilled in the art. See, for example, U.S. Patents No. 9,718,031; U.S. Patent No. 8,337,598; U.S. Patent No. 7,247,191; U.S. Patent No. 6,932,859 and U.S. Patent No. 6,755,900, all of which are incorporated herein by whole reference.

[0090] The membranes and fibers of the present invention are manufactured from a blend of proteins and polysaccharides. The protein-to-polysaccharide ratio is intended to be approximately 1:99 to approximately 99:1, approximately 1:10 to approximately 10:1, approximately 2:5 to approximately 5:2, approximately 3:7 to approximately 7:3, approximately 4:6 to approximately 6:4, or approximately 1:1, or any ratio within the range of ratios mentioned. In preferred embodiments, the protein content of the mixture is higher than the polysaccharide content. In preferred embodiments, the protein content is approximately 90%, 95%, 98%, 99%, or more.

[0091] The membrane of the present invention is further enhanced, i.e., given increased integrity and strength, but it is further intended that a manufacturing process step for crosslinking proteins in the membrane be incorporated. The inventors have found that, after the formation of the membrane of the present invention, when the membrane is exposed to an energy source at an appropriate energy level for an appropriate amount of time, the proteins are at least partially crosslinked, thereby giving the membrane of the present invention increased integrity compared to prior art membranes. The following illustrative section provides examples of several membranes (i.e., hollow fiber membranes) treated with and without heat or irradiation. Hollow fibers manufactured without the addition of exposure to the energy sources mentioned lacked integrity compared to hollow fibers manufactured with the addition of exposure to the energy sources.

[0092] Heat can be supplied via either dry heat or moist heat. One method of the present invention utilizes a temperature of about 60°C to about 100°C at a pressure of 0 psi (ambient pressure) to 20 psi or more for about 2 to about 60 minutes at a relative humidity of about 50% to 100%. Furthermore, heat can be supplied by immersing the membrane or fiber of the present invention in a water bath of about 60°C to about 100°C under atmospheric conditions.

[0093] The membranes and fibers of the present invention may also be exposed to energy through any form of radiation (e.g., electron beam, gamma, UV, etc.). The membranes and fibers of the present invention may be irradiated with about 1 to about 100 kGy, about 5 kGy to about 75 kGy, or about 10 kGy to about 50 kGy. The membranes and fibers of the present invention may be exposed to the radiation for about 0.1 minutes to about 60 minutes, about 1 minute to about 50 minutes, about 2 minutes to about 40 minutes, and about 2 minutes to about 30 minutes, and any value that falls within the listed values.

[0094] Hollow fiber manufacturing techniques, particularly membrane manufacturing techniques, are generally known to those skilled in the art (see, for example, Vandekar, VD, Manufacturing of Hollow Fiber Membrane, Int'l J Sci&Res, 2015, 4:9, pp.1990-1994, and the references cited therein). Similar to flat sheet membranes, known methods for manufacturing hollow fibers typically involve a certain phase separation technique. Common non-solvent-induced phase separation methods include thermally induced phase separation, vapor-induced phase separation, heat-induced phase separation (see, for example, U.S. Patent No. 5,444,097 of MilliporeSigma, incorporated herein by reference), or combinations thereof. However, other techniques, such as thermal extrusion and stretching, can be used for hollow fiber and membrane formation. Typically, these destabilize the polymer in solution by non-solvent, thermal destabilization, or removal of the solvent. As described herein, the dissolution of the polymer (in this case, polysaccharides and proteins) is followed by gelation or solidification through multiple crosslinking steps. The fibers can be further stretched to produce fibers having a diameter of less than 100 μm and a wall thickness of about 10 μm.

[0095] The membrane sheets can be manufactured using similar phase inversions in which a liquid polymer solution is immersed in a rapidly cooled solution and the solvent is withdrawn, causing the liquid polymer solution to solidify, as well as other techniques known to those skilled in the art, such as solvent evaporation (see, for example, U.S. Patent Application Publication No. 2020 / 0368696 of MilliporeSigma). See, for example, Gas Separation Membranes, Polymeric and Inorganic, Chapter 4, Ismail, et al., Springer, 2015 and U.S. Patent Application Publication No. 2007 / 0084788 of MilliporeSigma.

[0096] In some aspects of the present invention, pH-induced phase separation ("pH Induced Phase Separation" or "Proton Induced Phase Separation"; Satoru Tokutomi, Kazuo Ohki, Shun-ichi Ohnishi, Proton-induced phase separation in phosphatidylserine / phosphatidylcholine membranes, Biochimica et Biophysica Acta (BBA), Biomembranes, Volume 596, Issue 2, 28 February 1980, Pages 192-200) is used in the production of the membranes of the present invention (i.e., hollow fiber and sheet membranes). pH-induced phase separation is illustrated in the Examples section below. While pH-controlled liquid phase separation of polymers has been studied in cell physiology (Adame-Arana, O., et al., Liquid Phase Separation Controlled by pH, 2020 Oct 20;119(8):1590-1605; Epub 2020 Sep 16), the inventors believe they are the first to utilize pH-induced phase separation in the production of hollow fibers and sheet membranes, particularly membranes suitable for the production of clean meat or clean structured meat products. The use of pH-induced phase separation provides unexpected and remarkable benefits to the membranes of the present invention, namely, improved mechanical integrity, pore size, and porosity compared to conventional methods.

[0097] Dry spinning involves dissolving the polymer in a highly volatile solvent. The solvent / polymer mixture is then extruded and heated after the solvent evaporates, causing the polymer to solidify.

[0098] Wet spinning is more versatile because it involves a greater number of parameters that can be modified. The polymer-solvent mixture is extruded into a non-solvent bath where demixing and / or phase separation occur for the exchange of solvent and non-solvent. Between the extruded material and the non-solvent bath, there is a void where the formation of a hollow fiber membrane begins.

[0099] A technique that can eliminate or minimize the use of solvents is melt spinning with cold drawing (MSCS). This method offers cost-effective manufacturing but may come at the expense of structural control and the potential degradation of food materials. In this technique, the material is heated for extrusion and then stretched as it cools to mechanically form pores in the hollow fiber walls. All three of these techniques have been extensively studied and are well-known in the art (see Tan, XM. and Rodrigue, D., Polymers (Basel), 2019, Aug 5:11(8)).

[0100] Modifications of these techniques are also known to those skilled in the art. For example, see International Publication 2011 / 108929 (which is incorporated herein by reference in its entirety), which discloses a modified wet spinning extrusion method for producing hollow fibers composed of multiple polymers and polymer layers. The production of hollow fibers from non-synthetic materials is also known to those skilled in the art. For example, see U.S. Patent No. 4,824,569 of Suzuki, which is incorporated herein by reference in its entirety.

[0101] Hollow fiber membrane of the present invention for manufacturing structured meat products In one embodiment, the macroscopic structure of the hollow fiber of the present invention is intended to promote cell orientation along the fiber. In this regard, it is desirable by the present invention that the orientation of the component molecules that make up the hollow fiber be parallel, essentially parallel, or mainly parallel to the length of the hollow fiber. It is further intended that the component molecules create a surface texture, at least on the outer surface of the hollow fiber, that aids cell adhesion and cell orientation. Thus, in one embodiment, the surface texture of the hollow fiber of the present invention is intended to create adhesion points for cell adhesion. In another embodiment, it is further intended that cells grown on the hollow fiber of the present invention (in particular muscle cells, muscle cell-like cells, or cells having the characteristics of muscle cells) are similar to muscle cells in vivo and are oriented and elongated along the length of the hollow fiber, similar to muscle cells.

[0102] Therefore, the orientation of the scaffold surface structure directly correlates with the alignment of myotubes during formation. It can be thought of as if skeletal muscle wanted to form along an existing structure. It can be recalled that the fiber bundles closely mimic the skeletal muscle structure in order to form aligned myotubes. Thus, the hollow fiber bioreactor not only achieves tissue-like cell density but also myotube alignment that is not achieved by other techniques, giving it the most realistic mouthpiece of all the techniques discussed. The alignment phenomenon can be better understood by reviewing My mistake: Decellularized Apium graveolens Scaffold for Cell Culture and Guided Alignment of C2C12 Murine Myoblast - Santiago Campuzano, 2020, Ph.D. thesis, University or Ottawa, pp58-59.

[0103] With regard to the production of structured clean meat products, the hollow fibers of the present invention are intended to have a size range suitable for the present invention. The hollow fibers of the present invention are also intended to be spaced so that cells grown on the hollow fibers achieve a density similar to that of actual meat, with minimal void space between cells. In one embodiment, the hollow fibers of the present invention are intended to have an outer diameter of about 0.1 mm to about 3.0 mm, a porosity of about 0% (diffusion-based) to 75%, and a wall thickness of about 0.008 to about 0.5 mm or about 0.01 mm to about 0.2 mm, or any thickness between 0.008 mm and 0.5 mm that is not specifically repeated above. The inventors have found that this size is suitable for transporting culture medium through the lumen of the fiber, allowing for proper flow of culture medium through the walls of the hollow fibers, while being rigid enough to support cell growth and further providing the desired final product structure, texture, handling, and mouthfeel. However, other embodiments relating to variations in fiber diameter, wall thickness, and porosity are conceived depending on the desired structured clean meat product (e.g., beef, poultry, fish, pork, etc.) (described later).

[0104] Fiber Porosity The hollow fibers of the present invention need to have a porosity that allows for proper flow of culture medium through the fiber walls while simultaneously ensuring a surface suitable for cell proliferation and cell support. The porosity of the hollow fibers is partly related to the thickness of the hollow fiber walls and the composition of the hollow fibers. If the walls are sufficiently thin, a porosity of about 0% may be sufficient, allowing the culture medium to diffuse through the hollow fiber walls. The porosity of the hollow fibers of the present invention may be as high as 75% or 90%. Therefore, the range of porosity of the hollow fibers of the present invention is 0% to about 90%, about 10% to about 75%, about 30% to about 60%, or any percentage value between 0% and 75% that is not specifically repeated above.

[0105] The hollow fibers of the present invention may also undergo a pore formation process. The pore formation mechanism is one of the following techniques well known in the field of film formation: TIPS = thermally induced phase separation, NIPS = non-solvent-induced phase separation, VIPS = vapor-induced phase separation, pH-induced phase separation, MSCS = melt spinning combined with drawing (see Review on Porous Polymeric Membrane Preparation. Part II: Production Techniques with Polyethylene, Polydimethylsiloxane, Polypropylene, Polyimide, and Polytetrafluoroethylene, Xue Mei Tan, 1, 2, 2019). In all scenarios, the polymer becomes liquid by either thermal melting or chemical dissolution. From there, the polymer is extruded into a cylindrical shape and drawn onto a spindle. During the extrusion process, a bore fluid can be used to prevent the hollow fiber morphology from collapsing under its own weight. A pore formation chamber, such as a water bath or an atmospheric environment chamber, may also be present between the extrusion nozzle and the rewind spindle.

[0106] The present invention also intends to provide a configuration of the hollow fibers of the present invention in a bioreactor. The fiber configuration may include either or both the arrangement and / or spacing of the fibers. The fibers may be configured in any configuration that allows for the growth of a confluent cell population with minimal intercellular space. For example, the fibers can be oriented in a square / rectangle (rows and columns) or triangular / hexagon (honeycomb) packing mode. Thus, in one embodiment, the fibers are intended to be arranged to form a regular pattern of rows and columns when viewed from the edge. In another embodiment, the fibers are intended to form a honeycomb pattern when viewed from the edge. In another embodiment, the fibers of the present invention are intended to be arranged randomly or semi-randomly. In another embodiment, the hollow fibers are intended to be arranged in regular or semi-regular patterns of varying densities.

[0107] Hollow fibers can have outer diameters ranging from approximately 0.1 mm to 3.0 mm, 0.5 mm to 2.0 mm, and 0.8 mm to 1.3 mm, as well as any value between the cited values. A 1.0 mm hollow fiber is expected to have a flesh growth of approximately 0.3 mm to 0.5 mm around its outer diameter. An end diameter of approximately 1.1 mm can yield flesh of approximately 85 hollow fibers / cm².

[0108] In another embodiment, the fibers are intended to have varying degrees or amounts of interfiber spaces. For example, by using rows of higher-density fibers scattered among lower-density fibers, it is possible to generate variations in the texture of the final structured clean meat product, as is common in natural fish meat. Furthermore, it is intended that fibers of varying diameters, porosity, and wall thicknesses can be used in the same hollow fiber cartridge to simulate the appearance, texture, handling, and mouthfeel of natural meat.

[0109] In any configuration, fibers are spaced such that the distance between them allows sufficient flow of culture medium (and the nutrients, growth factors, etc. contained therein) to reach all of the cell aggregate. This is, of course, related, at least in part, to the flow rate of the culture medium and the porosity of the hollow fiber walls, but is largely related to the physical distance from the surface of the hollow fiber's outer wall to the cells. In other words, the culture medium and nutrients move or diffuse only over a limited distance through the cell aggregate. Currently, the maximum diffusion distance for oxygen and nutrients is considered to be 200 μm. Rouwkema, J., et al., (2009) Supply of Nutrients to Cells in Engineered Tissues, Biotechnology and Genetic Engineering Reviews, 26:1, 163-178. Therefore, the spacing between fibers should be approximately 400 μm from the outer wall of one fiber to the outer wall of an adjacent fiber. In culture conditions where the culture medium flows through both the hollow fibers and the spaces between them, the spacing can be larger. For example, the spacing can be 800 μm from the outer wall of one fiber to the outer wall of an adjacent fiber. These values ​​are for cases where the culture process relies solely on diffusion. However, the use of a pump (for example) allows for further separation of the fibers, creating a flow of medium from the hollow fibers through the cell culture space between the hollow fibers to the housing outlet (rather than relying solely on diffusion). For example, in some embodiments, the maximum distance between fibers is about 0.05 mm (50 μm) to about 5.0 mm; about 0.1 mm to about 3.0 mm; about 0.1 mm to about 2.0 mm; about 0.1 mm to about 1.0 mm or about 0.2 mm to about 0.5 mm, or any distance between the values ​​mentioned. While it is preferable in some embodiments for the medium to flow from the center of the hollow fibers through the culture to the housing outlet, the medium flow may be in the reverse direction, or may alternate between one direction and the other if desired. Alternating the direction of the medium flow is thought to help ensure that all cells receive an adequate supply of medium.

[0110] In one embodiment of the present invention, the degree of randomness is inherent to the distance between the hollow fibers of the present invention. The numerical values ​​given in the previous paragraph are the average inter-fiber distances for a given assembly. In one embodiment of the present invention, spacers and / or assembly techniques can be used to ensure, normalize, or control the inter-fiber distances. For example, see Han G, Wang P, Chung TS., Highly robust thin-film composite pressure retarded osmosis (PRO) hollow fiber membranes with high power densities for renewable salinity-gradient energy generation, Environ Sci Technol. 2013 Jul 16;47(14):8070-7. Epub 2013 Jun 28 or Chun Feng Wana, Bofan Li a, Tianshi Yang a, Tai-Shung Chung, Design and fabrication of inner-selective thin-film composite (TFC) hollow fiber modules for pressure retarded osmosis (PRO), Separation and Purification Technology, 172:32-42, 2017.

[0111] If the cell density becomes too high or the cell mass thickness becomes too thick, the culture medium will have difficulty reaching the cells furthest from the hollow fibers. Lack of culture medium for these cells can result in dead cells and / or dead space in the reactor where cells cannot grow. Consequently, the culture medium needs to flow through the hollow fiber cartridge to the housing outlet; that is, the flow of culture medium needs to be maintained at least until confluence is reached and the structured clean meat product is harvested. Those skilled in the art will be able to calculate the precise fiber spacing and porosity of the present invention for a given desired structured clean meat product based on the teachings herein.

[0112] The hollow fibers of the present invention can be arranged and fixed in what is referred to herein as a “hollow fiber cartridge.” In one embodiment, the hollow fiber cartridge is intended to be made by fixing the ends of the hollow fibers to end pieces in a desired arrangement. For example, each fiber has a first end and a second end. Each end is fixed to an end piece, i.e., a first and a second end piece. The end pieces may be, for example, a resin or plastic known in the art to be inert and non-toxic to cells. At least one of the first or second ends of the hollow fiber is positioned within the end piece such that the internal lumen of the hollow fiber is in fluid communication with the external environment. Thus, this arrangement of the hollow fiber within the end piece allows culture medium to flow from the external environment of the hollow fiber (i.e., outside the hollow fiber, but for example inside a sterile bioreactor) into the internal lumen of the hollow fiber.

[0113] Those skilled in the art understand how to assemble hollow fibers into modules or cartridges. These techniques are applicable to the hollow fibers of the present invention. Briefly, after spinning, the hollow fibers are cut to length, and the ends of the fibers are encased in a resin that flows around and solidifies (i.e., potted). Sometimes, the pores of the fibers can be sealed by encasing the cross-section of the fiber in a substance (e.g., calcined gypsum or other easily removable material known to those skilled in the art) to prevent the liquid resin from entering or blocking the pores of the fiber. See, for example, Vandekar, VD, Manufacturing of Hollow Fiber Membrane, Int'l J Sci&Res, 2015, 4:9, pp.1990-1994, and the references cited therein. In this invention, once the bundles are inserted into the housing for use in the production of the structured clean meat of the present invention, one or both ends of the "potted" bundles are trimmed or cut to expose the open ends of the fibers and allow the flow of the culture medium.

[0114] Furthermore, in some embodiments, the hollow fiber cartridge of the present invention is intended to have a fixing device for maintaining a desired distance between the first end piece and the second end piece. This may be necessary or preferred, for example, to facilitate insertion of the hollow fiber cartridge of the present invention into, for example, a bioreactor housing.

[0115] Accordingly, in one embodiment, the hollow fiber cartridge of the present invention is intended to contain a number of hollow fibers arranged in a desired configuration. The hollow fibers of the present invention have a first end and a second end. This configuration is maintained by fixing the first and second ends of the hollow fibers to a first end piece and a second end piece. When fixed as described, the hollow fibers are arranged parallel, substantially parallel, or essentially parallel to one another. Furthermore, the first end piece and the second end piece are arranged parallel, substantially parallel, or essentially parallel to one another. Moreover, the hollow fibers of the hollow fiber cartridge of the present invention are arranged perpendicular, substantially perpendicular, or essentially perpendicular to the end piece of the hollow fiber cartridge of the present invention. The diameter and length of the hollow fiber cartridge depend on the desired structured clean meat product and bioreactor configuration to be manufactured.

[0116] In one embodiment of the present invention, the hollow fibers of the hollow fiber cartridge of the present invention are approximately 40 to approximately 120 / cm². 2 The average density is approximately 60 to 100 / cm³. 2 Average density: approximately 70-90 / cm³ 2 The intended values ​​are the average density, or any value between the values ​​shown above that is not specifically repeated.

[0117] In one embodiment of the present invention, the hollow fibers in the hollow fiber cartridge of the present invention have void spaces between the hollow fibers before the addition of cells, and it is intended that the void spaces between the hollow fibers comprise about 25% to about 75% of the total area of ​​the hollow fiber cartridge, or about 40% to about 60% of the total area of ​​the hollow fiber cartridge, or any value between the above values ​​but not specifically repeated.

[0118] In one embodiment of the present invention, the hollow fiber cartridge of the present invention is intended to be designed to be removably inserted into a housing. That is, the cartridge can be inserted into the housing at the start of a production run and removed, i.e., harvested, at the end of the production run for any further desired processing of the structured clean meat product of the present invention. After harvesting the structured clean meat product, a new hollow fiber cartridge of the present invention can be inserted into the housing and the process can be repeated. In this regard, the housing for the hollow fiber cartridge of the present invention is part of a bioreactor or bioreactor system.

[0119] Reactor Configuration. The present invention is not limited to any particular reactor configuration or reactor system configuration, as long as sufficient medium flow can be maintained through the culture and the waste to be removed. Hollow fiber reactors are typically tubular in shape, but may be elliptical, flat (sheet-like), rectangular, or any other shape. In a preferred embodiment, the reactor includes an insertable / removable insert containing the hollow fibers of the present invention. After confluent cell growth (as defined herein) is reached, the insert can be removed, and the product can be completed by removing the insert ends and any further desired processing. Further processing may take the form of, for example, slicing, surface texture processing, flavoring, etc. Alternatively, further meat enhancement can be performed before harvesting and dismantling the apparatus. For example, the medium can be drained from the hollow fiber apparatus, and then additives will be introduced directly into or around the fibers.

[0120] Non-limiting examples of suitable reactor systems. The most suitable type of reactor system is a feed batch system, but any available reactor is intended to be suitable for use with the hollow fiber and hollow fiber cartridges of the present invention. For example, the MOBIUS(R) system (MilliporeSigma, Bedford, Massachusetts) is an example of a commercial system that can be readily converted for use in the present invention. Cells grown in another bioreactor may be seeded into the bioreactor in which the structured clean meat product is produced (i.e., the reactor containing the hollow fiber of the present invention). The bioreactor in which the hollow fiber apparatus is seeded (a reactor suitable for cell proliferation (growth) and cell expansion) may be an existing commercial reactor, such as a stirred tank or a corrugated reactor. The growth / expansion bioreactor may be, for example, a stirred tank or a corrugated reactor (known to those skilled in the art), and is intended to be a suspension, weak aggregate biomass, microcarrier culture, or other suitable reactor known to those skilled in the art. The manufacturing bioreactor (i.e., the reactor containing the hollow fibers of the present invention) is intended to be, for example, single-use, multi-use, semi-continuous, or continuous. The present invention further intends to provide a manifold for multiple reactors containing the hollow fibers of the present invention.

[0121] Accordingly, an exemplary reactor system of the present invention is intended to comprise one or more hollow fiber cartridges of the present invention, a housing sized to hold the hollow fiber cartridges; a culture medium supply source fluidized to one or more inlets within the housing; one or more culture medium outlets within the housing; and one or more pumps for supplying culture medium to the hollow fiber cartridges through the culture medium inlets and / or outlets, and / or for removing waste culture medium from the hollow fiber cartridges. Furthermore, the inlets are fluidized to the interior of the hollow fibers. Furthermore, the hollow fiber bioreactor may also comprise an automatic control device or automatic control system.

[0122] The present invention also relates to a method for producing meat products, for example, 100,000 to 100,000,000 cells (10 5 ~108 The present invention envisions a method comprising the steps of seeding one or more muscle cells, muscle-like cells, or cells designed to express one or more muscle-like characteristics at a density of ) into the void space between hollow fibers in the hollow fiber reactor of the present invention (Radisic, et al., Biotechnol Bioeng, 2003 May 20:82(4):403-414), and culturing the cells until a culture density of about 80% to about 99%, 85% to about 99%, 90% to about 99%, 95% to about 99%, 98% to about 99%, or about 100% (or any value between the listed percentage values) is achieved, and removing the first retaining device and the second retaining device from the first and second ends of the hollow fibers, respectively.

[0123] After seeding, the hollow fiber cartridge contains a culture medium supplied to the cells through one or both of the first and second ends of the hollow fibers into the interior of the hollow fibers, through the walls of the hollow fibers into the void spaces between the hollow fibers where the cells are seeded, and through one or more of the outlets into the housing. In another embodiment, it is intended that the culture medium may also flow between the fibers from both the inlet and outlet of the device. For example, one fluid path may pass through the fiber wall and a second fluid path may be around the fiber. It is intended that the device may have multiple inlets and outlets. After the cells have reached confluence, any residual culture medium and waste are flushed out, and one or more of the following are injected into the interior of the hollow fibers and / or any remaining void spaces between the cells: fats, flavors, colorants, salts, and preservatives.

[0124] Suitable fats for addition to the structured clean meat products of the present invention include, but are not limited to, saturated, monounsaturated, and polyunsaturated fats, such as corn oil, canola oil, sunflower oil, and safflower oil, olive oil, peanut oil, soybean oil, flaxseed oil, sesame oil, canola oil, avocado oil, seed oils, nut oils, safflower oil and sunflower oil, palm oil, coconut oil, omega-3, fish oil, lard, butter, processed animal fats, adipose tissue, or fats derived from cellular agriculture, or combinations thereof. Synthetic fats such as oleoresins can also be used. In fact, any fat recognized by the Food and Drug Administration (FDA) is suitable for use in the present invention and intended for use in the structured clean meat products of the present invention. The FDA Food Additives List includes natural substances and extracts (NATs), nutrients (NUTRs), essential oils and / or oleoresins (solvent-free) (ESOs).

[0125] Suitable flavors for use in the structured clean meat products of the present invention include, but are not limited to, any flavor listed on the FDA Food Additives List. These may be recorded as natural flavors (FLAV), essential oils and / or oleoresins (solvent-free) (ESO), enzymes (ENZ), natural substances and extracts (NAT), non-nutrient sweeteners (NNS), nutritional sweeteners (NUTRS), spices, other natural seasonings and flavors (SP), synthetic flavors (SY / FL), fumigants (FUM), artificial sweeteners including aspartame, sucralose, saccharin and acesulfame potassium, and yeast extracts, or combinations thereof, and are intended for use in the structured clean meat products of the present invention.

[0126] Suitable texture enhancers for use in the structured clean meat products of the present invention include, but are not limited to, refined plant materials, guar gum, cellulose, hemicellulose, lignin, beta-glucan, soybean, wheat, corn or rice isolates and beet fiber, pea fiber, bamboo fiber, plant-derived fiber, plant-derived gluten, carrageenan, xanthan gum, lecithin, pectin, agar, alginate and other natural polysaccharides, grain husks, calcium citrate, calcium phosphate, calcium sulfate, magnesium sulfate and salts, or any combination thereof, intended for use in the structured clean meat products of the present invention. These may be listed on the FDA Food Additives List as solubilizers and dispersants (SDAs) and natural substances and extracts (NATs).

[0127] Suitable nutritional additives for use in the structured clean meat products of the present invention include, but are not limited to, vitamins, trace elements, bioactive compounds, endogenous antioxidants such as vitamins A, B complex, C, D, and E, zinc, thiamine, riboflavin, selenium, iron, niacin, potassium, phosphorus, omega-3, omega-6, fatty acids, magnesium, proteins and protein extracts, amino acid salts, creatine, taurine, carnitine, carnosine, ubiquinone, glutathione, choline, glutathione, lipoic acid, spermine, anserine, linoleic acid, pantothenic acid, cholesterol, retinol, folic acid, dietary fiber, amino acids, and combinations thereof, all intended for use in the structured clean meat products of the present invention. Any food additive that is generally considered safe (GRAS) or approved by the FDA is intended for use in the structured clean meat products of the present invention and is incorporated herein. For example, see www.fda.gov / food / food-additives-petitions / food-additive-status-list.

[0128] Any natural or artificial food coloring that is generally considered safe (GRAS) or approved by the FDA is intended for use in the structured clean meat products of this invention. See, for example, www.fda.gov / industry / color-additive-inventories / color-additive-status-list.

[0129] Predictive Cell Types. The hollow fibers of the present invention are designed to be used for growing specific cell types suitable for the production of meat and meat products grown in vitro or in the laboratory, i.e., structured clean meat of the present invention. Thus, many different types of cells can be grown on the hollow fibers (and optionally in the hollow fiber cartridge of the present invention), but the fibers have been developed to be used for growing muscle cells (i.e., myocytes), or cells having the characteristics of muscle cells, or cells designed to have the characteristics of muscle cells (collectively referred to herein as muscle cells or myocytes) to confluence and to mimic the natural structure of muscle (i.e., meat). Preferably, the muscle is skeletal muscle. That is, the hollow fibers of the present invention are designed by the inventors to be suitable for growing muscle cells to obtain muscle fibers or myofibrils. Furthermore, other types of cells can be grown on the hollow fibers of the present invention and in reactors containing the hollow fibers of the present invention. These cells can be grown independently or in combination with muscle cells. For example, adipocytes or cells having or designed to have the characteristics of adipocytes (collectively referred to herein as adipocytes) can be cultured together with muscle cells to obtain a final product similar to natural muscle or flesh. The hollow fibers of the present invention are also suitable for co-culturing with other cells, such as fibroblasts, cells having the characteristics of fibroblasts, or cells designed to have the characteristics of fibroblasts.

[0130] In particular, with regard to the co-culture of muscle cells and adipocytes, the muscle cell to adipocyte ratio can be any ratio from 99:1, 95:5, 92:8, 90:10, 88:12, 85:15, 82:18, 80:20, 75:25, or 100:0 to 75:25 (including both ends).

[0131] Cells suitable for use in this invention can be obtained or induced from any animal from which food is currently obtained. Notable examples include cattle, pigs, sheep, fish (e.g., tuna, salmon, cod, haddock, shark, etc.), crustaceans, and birds (e.g., chickens, turkeys, ducks, etc.). More exogenous cell sources can also be used, such as animals that are traditionally hunted rather than farmed (e.g., deer, elk, moose, bears, rabbits, quail, wild turkeys, etc.) or combinations thereof.

[0132] The cells used in this invention can be induced by any method suitable for generating differentiated cells having desired properties. For example, any procedure suitable for inducing cells having differentiated muscle cell-like properties, adipocyte-like properties, etc. Such properties of muscle cells include, for example, having the appearance of long tubular cells and having large myosin and actin complements. Muscle cells also have the ability to fuse with other muscle cells to form myofibrils, which are muscle units that help give muscle, i.e., meat, its characteristic texture. Such properties of adipocytes (also called lipid cells and adipocytes in the art) include, for example, having large lipid cells that can occupy more than 90% of the cell volume, but are not limited to these. The hollow fibers of this invention provide, at least in part, a replacement for connective tissue (referred to in the art as "fascia") typically found in skeletal muscle.

[0133] Useful cells in the present invention include, but are not limited to, mesenchymal stem cells or cells derived from induced pluripotent stem cells (iPSCs). iPSCs are cells that have been manipulated to return to their pluripotent state, which can induce a number of cell types. In other words, iPSCs are pluripotent stem cells that can be directly produced from somatic cells. This technique was first reported in 2006 (Takahashi K, Yamanaka S, 25 August 2006, “Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors” Cell, 126(4):663-76), and has since advanced (see, for example, Li, et al., 30 April 2014, “Generation of pluripotent stem cells via protein transduction” Int. J. Dev. Biol., 58:21-27), and includes the production of muscle cells (see, for example, Rao, et al., 9 January 2018, “Engineering human pluripotent stem cells into a functional skeletal muscle tissue” Nat Commun., 9(1):1-12), and is well known to those skilled in the art.

[0134] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this invention pertains.

[0135] When introducing elements of the present disclosure or preferred embodiments thereof, the articles “a,” “an,” “the,” and “said” are intended to mean that there are one or more elements. The terms “comprising,” “including,” and “having” are intended to be comprehensive and mean that there may be additional elements other than those listed.

[0136] The transitional phrases “comprising,” “consisting essentially of,” and “consisting of” have the meanings set forth in MPEP 2111.03 (United States Patent Examination Manual, 9th Edition, revised October 2019; United States Patent and Trademark Office). Any claim using the transitional phrase “consisting essentially of” is understood to enumerate only the essential elements of the invention, and any other elements enumerated in any dependent claim are understood to be not essential to the invention enumerated in any dependent claim.

[0137] All ranges listed herein include all integers, fractions, and decimals, encompassing all values ​​within the cited range (including both endpoints). [Examples]

[0138] General materials and methods: All reagents were commercially available and used without further purification unless otherwise specified. Zein, sodium hydroxide, urea, hydroxypropylcellulose, k-carrageenan, sodium acetate, tripolyphosphate (TPP), (37% hydrochloric acid, antibiotic antifungal solution (100x dilution), and wheat gluten and bovine serum-derived fibronectin were purchased from MilliporeSigma (Burlington, Massachusetts). Bovine collagen was purchased from Corning (Corning, New York); soy protein isolate (SPI) was purchased from BulkSupplements (Henderson, Nevada); chitosan (mushroom-derived) was purchased from Modernist Panty (Elliott, Maine, USA); pea and peanut butter protein isolates were purchased from NorCal Organic (Crescent City, California); mung bean, broad bean, and chickpea protein isolates were purchased from Green Boy (Redondo Beach, California); agarose was purchased from Hispanagar (Burgos, Spain); and brown rice protein isolate was purchased from Zen Purchased from Principle (Incline Village, Nevada); sodium alginate and MooGloo® RM transglutaminase were purchased from Modernist Pantry (Elliott, Maine).

[0139] Cell culture stability test: The membranes were cut into 1 × 3.5-inch square samples and incubated in cell culture medium containing a 2x antibiotic antifungal solution (known to those skilled in the art) at 37°C for up to 21 or 30 days, depending on the experiment. For each membrane type, three samples were mechanically tested during incubation at each time point.

[0140] viscosity: Viscosity measurements were performed on doped solutions prepared using a Brookfield (Middleboro, Massachusetts) Viscometer DV-II+Pro with an S64 spindle.

[0141] Mechanical testing: Tensile tests were performed on 1 × 3.5 or 0.5 × 3.5 inch square samples using a Zwick Roell (Kennethaw, Georgia) TestControl II instrument, and the data were analyzed using Zwick Roell testXpert II V3.71 software.

[0142] Lyophilization: The samples were frozen in a scintillation vial under liquid nitrogen in water for 1 hour. The frozen samples were then dried using a Labconco (Kansas City, Missouri) freeze-dryer, 2.5 L, at 80°C.

[0143] Scanning electron microscopy: The sample is coated with 3 nm iridium, mounted on a stub, and imaged using a ThermoScientific (Waltham, Massachusetts) Quanta 200F or a JOEL (Peabody, Massachusetts) JCM 6000 scanning electron microscope (Tokyo, Japan).

[0144] Statistical analysis: The error bars are calculated as the standard error of the mean.

[0145] Rheology: Using a cone-shaped fixture, rheological analysis of the formulated dope and membrane was performed on a TA Ares rheometer (Newcastle, Delaware).

[0146] [Example 1] - Method for producing edible hollow fibers For schematic diagrams of exemplary manufacturing methods for producing the film of the present invention, please refer to Figures 1 and 2.

[0147] 1. Preparation of the dope solution a. The preparation of the dope solution requires a multi-step mixing process.

[0148] i. First, a protein solution was prepared. This required dissolving 14% by weight of plant protein concentrate in a weakly alkaline buffer. The mixture was homogenized at 20,000 rpm for several minutes. Specifically, micronized plant protein powder was used.

[0149] ii. The second solution contains a carrier polymer containing 2% alginate and 2% hydroxypropyl cellulose dissolved in the same buffer as the protein mixture. This was dissolved in a hybridizer at 35°C for 48 hours.

[0150] iii. The protein solution and the carrier polymer solution were mixed in a 1:1 ratio. Mixing was completed using an overhead stirring device, followed by 12 hours in a hybridizer at 35°C.

[0151] iv. The final mixture, having concentrations resulting from 2% polysaccharides and 7% plant protein, is called the doped solution.

[0152] b. The preparation of the bore solution is completed by dissolving 15 g / l calcium chloride in reverse osmosis (RO) water containing 0-1 g / l transglutaminase.

[0153] 2. Stretching and solidification a. Using a pressurized vessel and gear pump, the doping solution is forced through a coaxial orifice. There is a specific distance between the spinneret and the bath, which can be adjusted based on the rheological properties of the doping solution.

[0154] b. The solidification bath (also called the forming bath in this specification) is 15 g / l calcium chloride, which fixes the alginate to the 3D structure of the fiber by ion crosslinking.

[0155] 3.Crosslinking process a. In this application, the ionic crosslinking of the alginate may not adequately function in dissociating the divalent bonds from monovalent bonds formed by sodium salts in the cell culture medium. Crosslinking beyond that of the enzyme transglutaminase and alginate-calcium crosslinking was desired.

[0156] b. Next, the fibers were exposed to heat at nearly 100°C to thermally crosslink the proteins within the fibers. Proof of concept was demonstrated via autoclaving at 121°C for 60 minutes.

[0157] c. Alternatively or additionally, the fibers are exposed to electron beam or gamma ray irradiation of approximately 50 kGy (kilogray) to physically crosslink the cellulose portion of the mixture, i.e., to crosslink the proteins. As can be determined by those skilled in the art using the teachings herein, the final dose may be approximately 5 kGy to approximately 100 kGy, depending on the residence time of the material passing through the electron beam and the grade of the material.

[0158] 4. Coating process a. The fibers were passed continuously through a plasma chamber and then immersed in an aqueous solution of a 15% glycol / sorbitol (1:1) mixture (depending on the application, the glycol / sorbitol ratio may range from 1:14 to 14:1). This process was designed to minimize the breakdown of the porous structure of the hollow fibers via plasticizers.

[0159] Figures 3A and 3B show micrographs of hollow fiber membranes prepared by the method of Example 1. Figures 4A to 4C show scanning electron microscope images of hollow fiber membranes prepared by the method of this example. Figure 5A shows the length of a single hollow fiber prepared by the method of this example. Figure 5B provides a demonstration of the tensile strength of a single hollow fiber. [Example 2] - Predictive example of a fiber without a secondary crosslinking process a. Use the hollow fiber doped solution prepared as defined above in Example 1. Three conditions are covered in this example. All conditions are formed from the same doped solution. This doped solution consists of 1 part hydroxypropyl cellulose, 1 part sodium alginate (Sigma Aldrich, St. Louis, Missouri), and 7 parts pea protein isolate.

[0160] b. Under the first condition, the fibers are directly extruded into a 15 g / l calcium chloride bath and immediately solidified. After 10 minutes in the bath, the fibers are rinsed with MilliQ® water (MilliporeSigma, Bedford, Massachusetts) and then immersed in DMEM F12 medium for 72 hours. When the fibers are removed from the cell culture medium, they are unhandlable. The fibers can no longer support their own weight outside the solution. Most of the ionic crosslinking sites have dissociated.

[0161] c. In the second condition, the fibers are directly extruded into a 15 g / l calcium chloride bath and immediately solidified. After 10 minutes in the bath, the fibers are rinsed with MilliQ® water and then autoclaved at 121°C for 30 minutes. After cooling to room temperature, the fibers are immersed in DMEM / F12 (Dulbeccoo's Modified Eagle Medium / Nutrient Mixture F-12; ThermoFisher Scientific, Waltham, Massachusetts) medium for 72 hours. When the fibers are removed from the cell culture medium, they lose some degree of integrity. The fibers can be removed, but they can only self-support themselves to an extent of approximately 5 inches. Most of the ionic crosslinking sites are dissociated, but the thermally crosslinked proteins still play a role in enhancing the integrity of the fibers.

[0162] d. In the third condition, the fibers are directly extruded into a 15 g / l calcium chloride bath and immediately solidified. After 10 minutes in the bath, the fibers are rinsed with MilliQ® water and then exposed to a single pass of 50 kGy in a benchtop electron beam modifier and immersed in DMEM / F12 medium for 72 hours. Upon removal of the fibers from the cell culture medium, the fibers maintain their integrity and are able to support their own weight. Ionic crosslinking sites are readily dissociated in the cell culture medium, and some chain breaks may be present in both the alginate and cellulose backbones, but the physical crosslinking of the protein polymer network is resistant to dissociation in the medium.

[0163] These examples demonstrate that crosslinking of proteins by heat and / or irradiation results in improved integrity of the hollow fibers of the present invention, making them suitable for use, for example, in cell culture or filtration systems. [Example 3] - Preparation of dope solution 1.1. Protein Solution 1.1.2. Urea-based method: Zein: A zein solution (15% w / v) was prepared by adding 57 g of zein powder to 300 mL of MilliQ® water at 0°C under mechanical stirring. After 30 minutes, 11.25 g of urea was added to the suspension, followed by 83 mL of NaOH solution (0.4 N) (Figure 12). The reaction mixture was then heated to room temperature (23°C) and stirred for 18 hours before use.

[0164] Zein: A zein solution (19% w / v) was prepared by adding 72 g of zein powder to 300 mL of MilliQ® water at 0°C under mechanical stirring. After 30 minutes, 14.30 g of urea was added to the suspension, followed by 83 mL of NaOH solution (0.6 N) (Figure 12). The reaction mixture was then heated to room temperature (23°C) and stirred for 18 hours before use.

[0165] Zein-Hydroxypropylcellulose Blend: A 0.5% w / v hydroxypropylcellulose (HPC) solution (0.5% w / v) was prepared by adding 1.75 g of HPC to MilliQ® water and mixing by mechanical stirring for 18 hours. The solution was then cooled to 0°C using an ice bath, and 72 g of zein was added thereto. The suspension was stirred at 0°C for a further 20 minutes, after which 14.30 g of urea and 83 mL of NaOH solution (0.6 N) were added. The reaction mixture was heated to room temperature (23°C) and stirred for a further 18 hours before use.

[0166] Soy protein isolate: A soy protein isolate (SPI) solution (20% w / v) was prepared by adding 76 g of SPI powder to 300 mL of MilliQ® water under mechanical stirring. After 30 minutes, 11.25 g of urea was added to the suspension, followed by 83 mL of NaOH solution (0.4 N). The reaction mixture was then stirred for 18 hours before further use.

[0167] Pea protein isolate: A soy protein isolate (SPI) solution (20% w / v) was prepared by adding 76 g of PPI powder to 300 mL of MilliQ® water under mechanical stirring. After 30 minutes, 11.25 g of urea was added to the suspension, followed by 83 mL of NaOH solution (0.4 N). The reaction mixture was then stirred for 18 hours before further use.

[0168] Mung bean: A mung bean solution (15% w / v) was prepared by adding 57 g of PPI powder to 300 mL of MilliQ® water under mechanical stirring. After 30 minutes, 11.25 g of urea was added to the suspension, followed by 83 mL of NaOH solution (0.4 N). The reaction mixture was then stirred for 18 hours before further use. See Figure 6.

[0169] Wheat gluten: A gluten solution (15% w / v) was prepared by adding 56 g of gluten powder to 300 mL of MilliQ® water under mechanical stirring. After 30 minutes, 11.25 g of urea was added to the suspension, followed by 83 mL of NaOH solution (0.4 N). The reaction mixture was then stirred for 16 hours before further use.

[0170] 1.1.3. Hybridization-based methods: Mung bean alginate blend: 45 grams of mung bean protein isolate are weighed into 252 grams of water and homogenized at 25,000 rpm for 5 minutes to combine the mung bean protein isolate (Green Boy) and alginate (Modernist Pantry) blend. 3 mL of 10N NaOH (and optionally 6 g of urea) are then added and homogenized for another 5 minutes. The gel solution is then placed in a homogenizer at 40°C overnight.

[0171] 1.2. Alginate-protein blend solution Alternative mung bean and alginate dope formulations: Therefore, a ranging study was conducted to identify several possible formulations of the protein isolate and alginate. One exemplary formulation and mixing method is expressed by weight as follows: 0.2% alginate, 15% mung bean protein isolate, 1% 10N NaOH, 2% urea (optional), and 81.8% MilliQ® water.

[0172] The first step is to wet (i.e., suspend) and disperse the protein isolate in solution. Weigh the protein isolate and add MilliQ® water. Set a high-shear mixer, such as an IKA (Schönfen, Germany) homogenizer, to 25,000 rpm for 5-10 minutes, or until the slurry returns to fluid-like behavior. Once dispersed, add NaOH (and urea, if desired) to the protein and water, and then homogenize the solution for another 5 minutes until a viscous gel is formed. From there, stir the dissolved protein using an overhead mixer with a propeller set to 100-500 rpm. Slowly add the alginate to the mixture over 15 minutes. Once the alginate is homogeneously dispersed throughout the mixture and partially dissolved, transfer the solution to a jar, cover it, and place it in a hybridizer for 24 hours. See Figure 7.

[0173] i.1.2.1. Urea-based method Zein-Alginate: Zein solutions (15% w / v) prepared according to the urea method were mixed with pre-prepared alginate aqueous solutions of various concentrations (2% w / v, 4% w / v, 6% w / v, and 8% w / v) under mechanical stirring for 20 minutes to prepare zein-alginate blends with various biopolymer ratios.

[0174] SPI-Alginate: SPI solutions (20% w / v) prepared according to the urea method were mixed with pre-prepared alginate aqueous solutions of various concentrations (2% w / v, 4% w / v, 6% w / v, and 8% w / v) under mechanical stirring for 20 minutes to prepare SPI-alginate blends with various biopolymer ratios.

[0175] PPI-Alginate: PPI solutions (20% w / v) prepared according to the urea method were mixed with pre-prepared alginate aqueous solutions of various concentrations (2% w / v, 4% w / v, 6% w / v, and 8% w / v) under mechanical stirring for 20 minutes to prepare SPI-alginate blends with various biopolymer ratios.

[0176] Mung bean-alginate: Mung bean solution (15% w / v) prepared according to the urea method was mixed with pre-prepared alginate aqueous solutions of various concentrations (2% w / v, 4% w / v, 6% w / v, and 8% w / v) under mechanical stirring for 20 minutes to prepare mung bean-alginate blends with various biopolymer ratios.

[0177] Gluten-Alginate: Gluten solutions (15% w / v) prepared according to the urea method were mixed with pre-prepared alginate aqueous solutions of various concentrations (2% w / v, 4% w / v, 6% w / v, and 8% w / v) under mechanical stirring for 1 hour to prepare gluten-alginate blends with various biopolymer ratios.

[0178] 1.3. Protein-Agarose Blend Solution 1.3.1. Urea-based method Zein-Agarose: Zein-agarose blends with various biopolymer ratios were prepared by mixing a zein solution (15% w / v) prepared according to the urea method with pre-prepared agarose aqueous solutions of various concentrations (1% w / v, 2% w / v, and 4% w / v). Agarose solutions were prepared by adding each amount of agarose to 300 mL of MilliQ® water at 60°C and stirring for 2 hours until dissolution was complete. To obtain a homogeneous blend and avoid solidification of the agarose before casting, the freshly prepared agarose solution was added to a zein solution preheated to 40°C and stirred for 20 minutes. The solution was kept at 40°C before casting.

[0179] While we investigated zein-agarose, the formulation results and methods are expected to be similar to those for other plant-based proteins. Formulating agarose and maize protein (zein) is not a trivial task, as the two polymers do not utilize a common solvent or dissolution temperature. The combination of stabilizing zein above 40°C and reducing the required percentage of ethanol to less than approximately 20% is achieved simultaneously. Using the Minitab (Pennsylvania State University) design for experiments on formulations, we investigated solvent systems containing 0.04 N sodium hydroxide, urea, and ethanol in percentage w / v.

[0180] It was found that a combination of ethanol, urea, and 0.04N NaOH could dissolve zein. Surprisingly, zein could be dissolved with a 10% ethanol content in the presence of 0.04N NaOH and urea. The simplex design plot is shown in Figure 8. However, zein without ethanol was not stable at temperatures below approximately 40°C. This finding is supported by temperature-swept rheological data. See Figure 9.

[0181] Furthermore, this solvent system, consisting of approximately 5% urea, 19% ethanol, and 76% 0.04N NaOH, was found to reduce the gelation properties of agarose. See Figure 10.

[0182] Furthermore, when agarose and zein are mixed together in this solvent system, rheological properties are observed that demonstrate the feasibility of mixing both polymers together in a single system (see Figure 11).

[0183] Mung bean-agarose: Mung bean-agarose blends with various biopolymer ratios were prepared by mixing mung bean solution (15% w / v) prepared according to the urea method with pre-prepared agarose aqueous solutions of various concentrations (1% w / v, 2% w / v, and 4% w / v). Agarose solutions were prepared by adding each amount of agarose to 300 mL of MilliQ® water at 60°C and stirring for 2 hours until completely dissolved. To obtain a homogeneous blend and avoid solidification of the agarose before casting, the freshly prepared agarose solution was added to the mung bean solution preheated at 40°C and stirred for 20 minutes. The solution was kept at 40°C before casting.

[0184] Gluten-agarose: Various biopolymer ratios of gluten-agarose blends were prepared by mixing a gluten solution (15% w / v) prepared according to the urea method with pre-prepared agarose aqueous solutions of various concentrations (1% w / v, 2% w / v, and 4% w / v). Agarose solutions were prepared by adding each amount of agarose to 300 mL of MilliQ® water at 60°C and stirring for 2 hours until completely dissolved. To obtain a homogeneous blend and avoid solidification of the agarose before casting, the freshly prepared agarose solution was added to a zein solution preheated at 40°C and stirred for 20 minutes. The solution was kept at 40°C before casting.

[0185] 1.4 Plant-based chitosan Mushroom-based chitosan: Purchased from Modernist Pantry. Chitosan of various concentrations (5% w / v and 7% w / v) was dissolved overnight in 5% acetic acid via a hybridizer at 35°C. A forming bath containing 10 g / L triphenyl phosphate was used for solidification / crosslinking. The chitosan film was crosslinked overnight before processing.

[0186] 1.5K-Carrageenan K-Carrageenan: K-carrageenan was heated to 90°C in MilliQ® water at various concentrations (2% w / v, 4% w / v, and 10% w / v). At high temperatures, the solutions were cast onto a preheated plate and immersed in a forming bath containing 15 g / L calcium chloride. In another scenario, K-carrageenan was heated in solution with calcium chloride. Upon cooling, the solution solidified into a film.

[0187] b. Preparation / Formation of the film The films were cast using either an automatic film caster equipped with a 524-micron gap bar (BYK Drive 6 film caster, Reminster, Massachusetts) or a hand caster with a 600-micron gap. In both cases, 40 mL of doping solution was used for each film, approximately 25 × 15 cm. 2 The membrane dimensions were determined based on the area. Different coagulation conditions were applied depending on the membrane composition.

[0188] For hollow fibers, the doping solution was extruded through a coaxial needle purchased from Rame-hart Instrument, Co. (Sukkasana, New Jersey). Alternatively, a custom-made, laboratory-scale hollow fiber spinning machine was used, enabling processing at much higher viscosities (up to 100,000 centipoise: cP).

[0189] 2.1 Protein membrane Regardless of whether they were obtained by the urea method or the hybridizer method, flat sheet protein membranes were cast into sodium acetate buffer (0.2 M, pH 4.5) (see Figures 12A and 12B) and equilibrated in the buffer for 10 minutes to a maximum of 3 hours. The membranes were then washed with HEPES (0.1 M, pH 7.4) and stored in 70 / 30% w / v ethanol / aqueous solution.

[0190] In contrast, the zein membrane was stored in HEPES buffer (0.1 M, pH 7.4) containing twice the amount of antibiotic and antifungal solution.

[0191] 2.2 Protein-Alginate Blend Membrane Regardless of whether they were obtained by the urea method or the hybridizer method, flat-sheet protein-alginate blend membranes were cast into sodium acetate buffer (0.2 M, pH 4.5) containing CaCl2 (15 g / L) and equilibrated in the buffer for 10 minutes to a maximum of 3 hours. The membranes were then washed with HEPES (0.1 M, pH 7.4) containing CaCl2 (15 g / L) and stored in 70 / 30% w / v ethanol / aqueous solution.

[0192] Using doctor blade technique (as known to those skilled in the art), a mixture of alginate and mung bean protein is coated onto a PTFE sheet. The sheet is then placed in a pH 4.5 acetate buffer containing 15 g / L calcium chloride. The shift from pH 11 to pH 4.5 induces protein coagulation, and the calcium chloride crosslinks the alginate. The membrane is left in the buffer for 10 minutes on a bench. Once the membrane has formed and turned white (grayish-white), the membrane is removed and placed in a 99.5% glycerin bath with shaking for 10 minutes.

[0193] 2.3. Protein-agarose blend membrane Regardless of whether they were obtained by the urea method or the hybridizer method, protein-agarose blend membranes were cast from a hot solution maintained at 40°C into sodium acetate buffer (0.2 M, pH 4.5) and equilibrated in the buffer for 10 minutes to a maximum of 3 hours. The membranes were then washed with HEPES (0.1 M, pH 7.4) containing CaCl2 (15 g / L) and stored in 70 / 30% w / v ethanol / aqueous solution.

[0194] 3. Membrane crosslinking: 3.1 Transglutaminase (TG)-crosslinked protein-alginates Zein-Alginate-TG: The zein-alginate membranes prepared as described above were incubated for 24 hours at 4°C in MooGloo® solution (TG) (25% w / v) purchased from Modernist Pantry (Elliott, Maine), containing HEPES (0.1M, pH 7.4) and CaCl2 (15 g / L). 125 mL of MooGloo® solution was used for each membrane. Subsequently, each membrane was washed twice with 250 mL of HEPES (0.1M, pH 7.4) containing CaCl2 (15 g / L). Finally, the membranes were stored in HEPES (0.1M, pH 7.4) containing CaCl2 (15 g / L), 2x concentrated penicillin-streptavidin, and an antifungal agent.

[0195] PPI-Alginate-TG: The PPI-alginate membranes prepared as described above were incubated at 4°C for 24 hours in a MooGloo® (TG) solution (25% w / v) containing HEPES (0.1M, pH 7.4) and CaCl2 (15 g / L). 125 mL of MooGloo® solution was used for each membrane. Subsequently, each membrane was washed twice with 250 mL of HEPES (0.1M, pH 7.4) containing CaCl2 (15 g / L). Finally, the membranes were stored in 70 / 30% w / v ethanol / aqueous solution.

[0196] Brown rice-alginate-TG: The brown rice-alginate membranes prepared as described above were incubated at 4°C for 24 hours in a MooGloo® (TG) solution (25% w / v) containing HEPES (0.1M, pH 7.4) and CaCl2 (15 g / L). 125 mL of MooGloo® solution was used for each membrane. Subsequently, each membrane was washed twice with 250 mL of HEPES (0.1M, pH 7.4) containing CaCl2 (15 g / L). Finally, the membranes were stored in a 70 / 30% w / v ethanol / aqueous solution.

[0197] Mung bean-alginate-TG: The mung bean-alginate membranes prepared as described above were incubated at 4°C for 24 hours in a MooGloo® (TG) solution (25% w / v) containing HEPES (0.1M, pH 7.4) and CaCl2 (15 g / L). 125 mL of MooGloo® solution was used for each membrane. Subsequently, each membrane was washed twice with 250 mL of HEPES (0.1M, pH 7.4) containing CaCl2 (15 g / L). Finally, the membranes were stored in 70 / 30% w / v ethanol / aqueous solution.

[0198] 3.2 Thermal crosslinking with glycerol 3.2.1 Protein membrane As water is exchanged throughout the porous structure, the membrane changes from translucent to transparent. The membrane is then removed and placed in a third bath set to 130°C for 10 minutes. Once the proteins have cross-linked, the membrane is placed in a final bath containing HEPES buffer at pH 7.4 to ensure the scaffold is at a physiological pH for biological performance.

[0199] SPI: SPI flat sheet membranes were cast into sodium acetate buffer (0.2 M, pH 4.5) using a PTFE support sheet and equilibrated in the buffer for 10 minutes to a maximum of 3 hours. The PTFE-supported membranes were then transferred to a glycerol bath, and the aqueous solution was replaced with glycerol for 10 minutes to 3 hours. The membranes were then thermally crosslinked by passing them through a hot glycerol bath or by using an oven. In the first case, the membranes were transferred to a 100°C stirred glycerol bath and incubated for 10 minutes. Various temperature gradients were then investigated by varying the final temperature (between 110°C and 140°C) and temperature increments of the glycerol bath. In the case of oven treatment, the membranes were incubated at various temperatures in the range of 100°C to 140°C and for various durations of 10 to 24 hours.

[0200] Mung bean: Mung bean flat sheet membranes were cast into sodium acetate buffer (0.2 M, pH 4.5) using a PTFE support sheet and equilibrated in the buffer for 10 minutes to a maximum of 3 hours. The PTFE-supported membranes were then transferred to a glycerol bath, and the aqueous solution was replaced with glycerol for 10 minutes to 3 hours. The membranes were then thermally crosslinked by passing them through a hot glycerol bath or by using an oven. In the first case, the membranes were transferred to a 100°C stirred glycerol bath and incubated for 10 minutes. Various temperature gradients were then investigated by varying the final temperature (between 110°C and 140°C) and temperature increments of the glycerol bath. In the case of oven treatment, the membranes were incubated at various temperatures in the range of 100°C to 140°C and for various durations of 10 to 24 hours.

[0201] Wheat gluten: Wheat gluten flatsheet membranes were cast into sodium acetate buffer (0.2 M, pH 4.5) using a PTFE support sheet and equilibrated in the buffer for 10 minutes to a maximum of 3 hours. The PTFE-supported membranes were then transferred to a glycerol bath, and the aqueous solution was replaced with glycerol for 10 minutes to 3 hours. The membranes were then thermally crosslinked by passing them through a hot glycerol bath or by using an oven. In the first case, the membranes were transferred to a 100°C stirred glycerol bath and incubated for 10 minutes. Various temperature gradients were investigated by varying the final temperature of the glycerol bath (between 100°C and 140°C). In the case of oven treatment, the membranes were incubated at various temperatures in the range of 100°C to 140°C and for various durations of 10 to 24 hours.

[0202] 3.2.2 Protein-Alginate Membrane Mung bean-alginate: Mung bean-alginate flat sheet membranes were cast into sodium acetate buffer (0.2 M, pH 4.5) containing CaCl2 (15 g / L) using a PTFE support sheet and equilibrated in the buffer for 10 minutes to a maximum of 3 hours. The PTFE-supported membranes were then transferred to a glycerol bath, and the aqueous solution was replaced with glycerol for 10 minutes to 3 hours. The membranes were then thermally crosslinked by passing them through a hot glycerol bath or by using an oven. In the first case, the membranes were transferred to a 100°C stirred glycerol bath and incubated for 10 minutes. Various temperature gradients were then investigated by varying the final temperature (between 110°C and 140°C) and temperature increments of the glycerol bath. In the case of oven treatment, the membranes were incubated at various temperatures in the range of 100°C to 140°C and for various durations of 10 to 24 hours. See Figure 13.

[0203] Wheat gluten-alginate: Wheat gluten-alginate flat sheet membranes were cast into sodium acetate buffer (0.2 M, pH 4.5) containing CaCl2 (15 g / L) using a PTFE support sheet and equilibrated in the buffer for 10 minutes to a maximum of 3 hours. The PTFE-supported membranes were then transferred to a glycerol bath, and the aqueous solution was replaced with glycerol for 10 minutes to 3 hours. The membranes were then thermally crosslinked by passing them through a hot glycerol bath or by using an oven. In the first case, the membranes were transferred to a 100°C stirred glycerol bath and incubated for 10 minutes. Various temperature gradients were then investigated by varying the final temperature (between 110°C and 140°C) and temperature increments of the glycerol bath. In the case of oven treatment, the membranes were incubated at various temperatures in the range of 100°C to 140°C and for various durations of 10 to 24 hours.

[0204] Zein-Alginate: Zein-alginate flat sheet membranes were cast into sodium acetate buffer (0.2 M, pH 4.5) containing CaCl2 (15 g / L) using a PTFE support sheet and equilibrated in the buffer for 10 minutes to a maximum of 3 hours. The PTFE-supported membranes were then transferred to a glycerol bath, and the aqueous solution was replaced with glycerol for 10 minutes to 3 hours. The membranes were then thermally crosslinked by passing them through a hot glycerol bath or by using an oven. In the first case, the membranes were transferred to a 100°C stirred glycerol bath and incubated for 10 minutes. Various temperature gradients were then investigated by varying the final temperature of the glycerol bath (between 100°C and 110°C and 100°C and 140°C). In the case of oven treatment, the membranes were incubated at various temperatures in the range of 100°C to 140°C and for various durations of 10 to 24 hours.

[0205] 4. Film coating 4.1 Bovine collagen coating (Method 1) Mung bean membranes were coated with bovine collagen to increase their affinity for cells, thereby promoting cell adhesion and proliferation. Dry 14 mm diameter mung bean membrane discs were immersed in a 3 mg / mL collagen solution at room temperature for 2 hours (20 discs per 20 mL of collagen solution). The collagen solution was then removed, and the discs were placed in a 100% ethanol solution and stored at 4°C before use.

[0206] 4.2 Bovine collagen coating (Method 2) Mung bean membranes were coated with bovine collagen to increase their affinity for cells and promote cell adhesion and proliferation. Dry 14 mm diameter mung bean membrane discs were immersed in a 3 mg / mL collagen solution at room temperature for 2 hours (20 discs per 20 mL of collagen solution). The collagen solution was then removed, and the discs were incubated in HEPES solution (0.1 M, pH 7.4) at 37°C for 1 hour. The HEPES solution was then removed, and the discs were stored in a 70 / 30 w / v ethanol-aqueous solution at 4°C before use.

[0207] 4.3 Bovine fibronectin coating (Method 1) Mung bean membranes were coated with bovine fibronectin to increase their affinity for cells, thereby promoting cell adhesion and proliferation. Dry 14 mm diameter mung bean membrane discs were immersed in a 2.5 mg / mL fibronectin solution at room temperature for 2 hours (20 discs per 20 mL of fibronectin solution). The fibronectin solution was then removed, and the discs were placed in a 100% ethanol solution and stored at 4°C before use.

[0208] 4.4 Chitosan coating Mung bean membranes were coated with chitosan to increase their affinity for cells and promote cell adhesion and proliferation. Dry 14 mm diameter mung bean membrane discs were immersed in a 1% w / v chitosan acetate solution at room temperature for 1 hour (20 discs per 20 mL of 0.2 M, pH 4.5 chitosan solution). The chitosan solution was then removed, and the discs were placed in a 10% TPP solution and stirred for 3 hours. The discs were then washed twice with MilliQ® water and stored at 70 / 30 w / v at 4°C.

[0209] keep: The membranes can be stored in 70 / 30 ethanol / MilliQ(trademark) w / v OR HEPES containing an antibiotic / antifungal agent. Even if drying is possible, care must be taken to avoid pore breakdown. Drying can be achieved using a freeze-drying apparatus. More scalable and flexible membranes can be dried by changing the HEPES using a separate exchange bath consisting of water and 20-40% glycerin. A porous structure can be dried if the membrane pores are filled with 20-40% glycerin. See Figure 38.

[0210] 5. Mechanical testing of the film The mechanical properties of the membranes were characterized in tensile mode using a ZwickRoel testing machine. As shown in Figure 14, the membrane moduli cover a wide range of values, enabling our material portfolio to comprehensively address diverse design specifications for hollow fibers. For example, the k-carrageenan-based membrane has a moduli of less than 100 kPa and is therefore suitable as a substrate for muscle cell proliferation and differentiation (see Figure 15). Since the hollow fibers become part of the final cultured meat product, the texture profile of actual meat also needs to be considered as a design specification for our materials. In this regard, we designed heat-treated soy, agarose blends and some alginate blends that fall within the moduli range of 100-300 kPa, which is known to be characteristic of meat, particularly whole-cut steaks. The highest mechanical performance in terms of moduli and fracture strain is achieved with pure proteins such as mung bean and zein, or alginate-protein blends. These final materials can be used as structural components that allow the hollow fibers to maintain working conditions once they have gone through a wide range of manufacturing processes and are finally installed in a bioreactor.

[0211] result 5.1 Optimization of the Glycerol Method The final step of glycerol crosslinking, including sequential coagulation (1), neutralization (2), glycerol-water exchange (3), and glycerol heat treatment (4), was verified by testing the effect of each step shown in Table 1. In the absence of glycerol heat treatment after coagulation in acetate and neutralization in HEPES (Sample 1, AC-H-0-0), a mechanically unstable film with paste-like consistency was obtained (see Figure 16). Similarly, replacing the glycerol treatment with autoclaving (121°C) resulted in an unstable and brittle film (Sample 4 AC-0-0-HW). Even when the initial acetate coagulation and neutralization steps were removed and only glycerol heat treatment was applied (Sample 3 0-0-G-HG), a powdery, mechanically unstable film was obtained (see Figure 16). This highlights the importance of having a coagulation protein network essential for film stability. Furthermore, when coagulation occurs under neutral conditions (HEPES) rather than acidic conditions, a very brittle film is obtained (Sample 4 0-HG-HG). Finally, replacing water with glycerol at room temperature before heat treatment helps avoid the formation of large bubbles due to the sudden expansion of water when in contact with a heated glycerol bath (Sample 5 AC-0-0-HG). As a result, the best film was obtained by coagulating the doped solution using an acetate bath, replacing water with glycerol at room temperature, and finally thermal crosslinking the protein network using a heated glycerol bath (Sample 6 AC-0-G-HG). Compared with other film samples, those obtained according to AC-0-G-HG resulted in the most stable film with the highest Young's modulus and lowest strain, and exhibited a higher degree of protein crosslinking (see Figure 17).

[0212] [Table 1] Table 1: Experimental conditions for optimizing the glycerol crosslinking method. AC represents "0.2 M acetate bath at pH 4.5", H represents "0.1 M HEPES bath at pH 7.4", G represents "glycerol bath", HG represents "hot glycerol bath", HW represents "hot water treatment" (autoclave 121°C), 0 represents "do not perform the process", Y represents "yes", and N represents "no".

[0213] Each step of the glycerol-based heat treatment was further optimized to improve the morphology and mechanical properties of the film. The effect of the acetate solidification process was investigated by varying the duration of the acetate bath and keeping both the water-glycerol exchange (10 minutes) condition and the glycerol-based heat treatment (temperature gradient: 10 minutes at 100°C, gradient to 120°C, and 30 minutes at 120°C) condition constant. Figure 18 shows the mechanical properties of films solidified for 10 minutes to a maximum of 3 hours. No statistical differences in elastic modulus, final strain, and final stress were observed even with increased solidification time, indicating that solidification was completed within the investigated 10-minute timeframe. These results suggest that 10 minutes is sufficient to neutralize the film pH and therefore enable the successful solidification process. Next, the glycerol-based heat treatment was investigated by keeping both the duration of the solidification bath (10 minutes) and the water-glycerol exchange (10 minutes) constant and varying the duration of the heat treatment after reaching the final temperature of 120°C. As shown in Figure 19, increasing the heat treatment time results in stronger and tougher films, with final strain and stress values ​​doubling and tripling, respectively. After 30 minutes, the mechanical properties of the films begin to plateau, and it is also noted that there is little difference in final stress between the 30-minute and 60-minute samples. Since heat treatment appears to have a greater effect on the mechanical properties of the films, further investigations were conducted to evaluate the effect of the final temperature gradient. Here, rheological analysis was used to monitor changes in the physical properties of the films. Films that had been initially solidified (10 minutes) and subjected to water-glycerol exchange (10 minutes) were directly heat-treated in a rheometer chamber. As shown in Figures 20 and 21, the films were subjected to a 4°C / min heat gradient starting at 20°C and equilibrated at three different final temperatures: 100°C, 120°C, and 140°C. At 50-60°C, the tangent (δ) began to decrease, thus suggesting that the initiation of the protein annealing process leads to film solidification. The solidification process is thought to be driven by heat-induced protein unfolding and the formation of interchain physical crosslinks. Interestingly, a tangent (δ) trend is observed when the final temperature of the isothermal gradient changes.Lower values ​​of the tangent (δ) were obtained over the increasing final temperature gradient, thus suggesting that the film undergoes a strengthening process as the annealing temperature increases. This trend was confirmed by tensile tests performed on samples obtained from rheological experiments. As shown in Figure 20, increases in modulus, final stress, and strain are observed as the final isothermal temperature increases.

[0214] The formation of the membrane structure begins around 50-60°C and continues at the same rate regardless of the final isothermal temperature. However, the final strength of the resulting membrane structure appears to be influenced by the final isothermal conditions. Higher isothermal temperatures result in membranes with higher elasticity (lower tangent (δ)).

[0215] 6. Stability test in cell culture medium To test the stability of the materials under cell culture conditions, the membranes were incubated in cell culture medium at 37°C for up to 30 days, and mechanical tests were performed at various time points to investigate their integrity. The k-carrageenan and its pea protein isolate blend proved to be highly unstable in cell culture medium, having already completely dissolved after one day of incubation. In contrast, the alginate and agarose blends were found to be more stable over longer incubation times. In the latter case, the performance of the membrane is thought to be primarily influenced by the stability of the alginate and agarose polysaccharide components. This finding is supported by the presence of two different stability trends depending on the properties of the polysaccharides. The alginate blend underwent a dramatic decrease in both modulus and strain, while the zein blend underwent a decrease in modulus of more than 10 times. In contrast, the agarose blend maintained its mechanical properties almost perfectly throughout the entire 21-day incubation period. See Figures 22, 23, 24, and 25.

[0216] In the case of alginate blends, the gradual decrease in mechanical stability was thought to be caused by the decomposition of the calcium-glutaric acid crosslinked polymer network. This hypothesis was supported by the remarkable swelling behavior of the membrane during incubation time, which was quantified as an increase in membrane surface area (Figure 22). In contrast, no swelling was observed in agarose blend-based membranes. The correlation between the tendency to swell and the tendency to maintain mechanical stability indicates that the polysaccharide network is the main structural component of the membrane that underwent disintegration under culture conditions in the case of alginate.

[0217] To enhance the stability of the alginate-protein blend under cell culture conditions, we investigated crosslinking of the protein components. Transglutaminase was selected as the primary crosslinking candidate for testing, as it is commonly used in the food industry to prepare processed meats. In this case, as shown for the brown rice-alginate blend, a decrease in both elastic modulus and strain was observed with increasing incubation time. See Figure 26.

[0218] Thermal annealing was selected as an alternative method to induce physical crosslinking of protein polymer networks and ultimately stabilize the membrane during cell culture. To avoid the collapse of the porous membrane structure formed by the reverse transition, glycerol was used as both a water exchange medium and a heat transfer vector for the annealing process. Compared to alginate blends, both thermally annealed soybean and mung bean membranes did not show a decrease in elastic modulus when incubated in cell medium at 37°C. After 21 days, the soybean membrane showed an increase in elastic modulus, nearly doubling in value. Breaking strain (elongation at break) was unaffected, but in the case of soybean, a slight decrease in surface area suggested further crosslinking processes that may occur over time. After 30 days of incubation, a slight decrease in the force required to cause breakage was observed for mung bean membranes. Due to their higher mechanical stability under cell culture conditions compared to alginate-protein blends and higher breaking strain compared to agarose-protein blends, these thermally treated pure protein materials are preferred candidates for developing membranes for bioreactor applications. Please refer to Figure 27.

[0219] 7. Imaging of porosity 7.1 Flat Sheet Film The porosity of the fabricated membranes was investigated via scanning electron microscopy. As shown in Figures 28 and 29, respectively, the heat-treated soybean and mung bean protein membranes exhibit heterogeneous porosity, characterized by smaller pores in the submicron range on the surface and larger pores in the 20–50 micron range located in the cross-section. The rapid coagulation process occurring at the membrane-bath solution interface during the coagulation process is thought to be the origin of the thinner porosity located on the surface. In contrast, the slower coagulation process occurring in the membrane core allows for greater phase separation resulting in larger pores. In the case of zein and agarose-zein, a different scenario was observed, with homogeneous porosity observed throughout the membrane. Figure 30 shows that in this latter case, the phase separation process was the result of a fibrillation process that resulted in a very homogeneous pore size distribution. Although the present invention is not limited by theory, it is assumed that both agarose and zein are known to undergo fibrillation via protein self-assembly. Similar results were observed in the alginate-zein and pea-k-carrageenan membranes (see Figure 31), indicating that fibrillation of the biopolymer was a major step in membrane formation. In contrast, a skinning effect was observed in the mung bean-agarose and soybean-alginate membranes. See Figure 32.

[0220] 7.2 Hollow Fiber Membrane The porosity of the hollow fibers was investigated using a scanning electron microscope. Figure 33 shows the cross-section (top) and surface (bottom) of mung bean-alginate (15%-0.2%) hollow fibers. The fibers exhibited pores of less than 50 microns throughout the entire cross-section, but no skinning effect was observed. The fiber wall thickness was in the 100 micron range, a value aimed at optimizing external nutrient diffusion, considering theoretical diffusion typically observed in tissues with a thickness exceeding 200 microns.

[0221] 8. Cell adhesion and proliferation test The fabricated membranes were tested for cell adhesion and proliferation using C2C12 (see Figures 34, 35, and 36) and QM7 (see Figure 37) cell lines. In general, pure protein membranes yielded higher adhesion and proliferation, a finding supported by the presence of more elongated cells in both C2C12 and QM7 cells. The best results were achieved when the protein membranes were coated with cell adhesion proteins such as collagen and fibronectin. In contrast, protein-polysaccharide blends resulted in more spherical, cluster-like aggregated cells, indicating low affinity of the material to both C2C12 and QM7 cell lines.

Claims

1. A method for producing edible cross-linked porous hollow fibers or sheet membranes, a) i) a step of preparing one or more edible proteins, ii) one or more solvents, and iii) a forming bath; wherein the one or more solvents or the forming bath also includes one or more polyvalent cations or anions or buffers; b) A step of co-mixing the one or more edible proteins in the one or more solvents to form a mixture; c) A step of extruding the mixture into the forming bath to form extruded hollow fibers, or casting the mixture into the forming bath to form a sheet film; d) A step of forming an edible crosslinked porous hollow fiber or sheet membrane by exposing the extruded hollow fiber or sheet membrane to an energy source selected from one or more heat and irradiation sufficient to at least partially crosslink the one or more proteins; A method that includes this.

2. The method according to claim 1, further comprising preparing one or more edible polysaccharides, and in step b), co-mixing the one or more polysaccharides with the one or more edible proteins in the one or more solvents.

3. The method according to claim 1, further comprising preparing a plasticizer and co-mixing the plasticizer with the one or more edible proteins in the one or more solvents in step b).

4. The method according to claim 1, wherein one or more of the proteins are selected from the group consisting of peas, soybeans, wheat, pumpkin, rice, brown rice, sunflower, canola, chickpeas, lentils, mung beans, white beans, corn, oats, potatoes, quinoa, sorghum, and peanuts.

5. The method according to claim 2, wherein the one or more polysaccharides are selected from the group consisting of agar, chitosan, chitin, alginate, sodium alginate, cellulose, hydroxypropylcellulose, methylcellulose, hydroxypropylmethylcellulose, gellan gum, xanthan gum, pectin, tapioca, guar gum, and bean gum.

6. The method according to claim 1, wherein the one or more solvents are selected from the group consisting of water, acetic acid, citric acid, lactic acid, phosphoric acid, malic acid, tartaric acid, sodium hydroxide, ethanol, glycerin, and propylene glycol.

7. The method according to claim 1, wherein the forming bath contains one or more of the following in combination with 1) water, acetic acid, citric acid, lactic acid, phosphoric acid, malic acid, and tartaric acid, or 2) one or more of sodium hydroxide and potassium hydroxide, which in turn contain one or more of calcium, zinc, magnesium, iron, and potassium.

8. The method according to claim 1, wherein the ion is selected from the group consisting of Ca2+, Mg2+, Fe3+, Zn2+, tripolyphosphate, and trisodium citrate, and the selected ion can at least enable partial crosslinking of the one or more polysaccharides.

9. The method according to claim 1, wherein the heat in step d) is approximately 70°C to approximately 140°C, applied under a pressure of approximately 0 PSI to approximately 20 PSI gauge and a relative humidity of approximately 50% to approximately 100%, for approximately 2 to approximately 60 minutes, or the hollow fiber or sheet membrane is immersed in a water bath at approximately 60°C to approximately 100°C under atmospheric conditions.

10. The method according to claim 1, wherein the mixture in step b) is heated.

11. The method according to claim 1, wherein the co-mixing in step b) is carried out at a temperature of approximately 0°C to approximately 90°C.

12. The method according to claim 1, wherein the mixture has a pH of about 10 to about 13, and the compounded bath has a pH of about 3 to about 5.

13. The method according to claim 12, wherein, after formation, the film is neutralized to a pH of approximately 6.8 to approximately 7.

8.

14. The method according to claim 12, wherein, after formation, the film is neutralized to a pH of approximately 7.3 to approximately 7.

5.

15. The method according to claim 1, wherein the irradiation is selected from the group consisting of electron beam, UV light, and gamma ray irradiation.

16. The method according to claim 15, wherein the irradiation is applied during or after the process.

17. The method according to claim 15, wherein the irradiation is approximately 1 to approximately 100 kGy or approximately 10 to approximately 50 kGy.

18. The method according to claim 1, wherein the porosity is approximately 1% to approximately 90%.

19. The method according to claim 1, wherein the porosity is approximately 50% to approximately 80%.

20. The method according to claim 1, further comprising the step of coating the edible crosslinked porous hollow fiber or sheet membrane with a coating to enhance cell adhesion.

21. The method according to claim 20, wherein the coating is selected from one or more of fibronectin, fibrinogen, laminin, collagen, gelatin, or short-chain peptide sequences isolated from these proteins.

22. The method according to claim 21, wherein the short-chain peptide sequence is selected from one or more of the group consisting of RGD, YIGSR, IKVAV, DGEA, PHRSN, and PRARI.

23. The method according to claim 1, further comprising the step of modifying the outer surface of the edible crosslinked porous hollow fiber to enhance cell adhesion.

24. The method according to claim 1, further comprising the step of coating the edible crosslinked porous hollow fiber or sheet membrane with a plasticizer.

25. The method according to claim 23, wherein the surface modification is selected from one or more of plasma, corona, abrasion, etching, ablation, or sputter coating.

26. The method according to claim 1, wherein the protein is powdered or finely ground before being dissolved in the solvent.

27. The method according to claim 1, wherein the protein is at least 70%, 80%, 90%, 95%, 98%, 99%, or 99.9% pure.

28. The method according to claim 1, wherein the polysaccharide is at least 70%, 80%, 90%, 95%, 98%, 99%, or 99.9% pure.

29. The method according to claim 1, wherein the ratio of protein to polysaccharide in the mixture is approximately 10:1 to approximately 1:10 or approximately 1:99 to approximately 99:

1.

30. The method according to claim 1, wherein the ratio of protein to polysaccharide in the mixture is approximately 4:1 to approximately 1:

4.

31. The method according to claim 1, wherein the ratio of protein to polysaccharide in the mixture is approximately 1:1 or approximately 7:

1.

32. The method according to claim 1, wherein the forming bath contains, in combination with i) one or more of water, acetic acid, citric acid, lactic acid, phosphoric acid, malic acid, and tartaric acid, or ii) one or more of sodium hydroxide and potassium hydroxide, one or more of calcium, zinc, magnesium, iron, and potassium.

33. A hollow fiber or sheet membrane prepared by any of the methods described in claims 1 to 32.

34. A method for producing edible cross-linked porous hollow fibers or sheet membranes, a) i) one or more edible proteins, ii) one or more edible polysaccharides, iii) one or more solvents, and iv) a forming bath, wherein the forming bath contains one or more of the following in combination with 1) water, acetic acid, citric acid, lactic acid, phosphoric acid, malic acid, and tartaric acid, or 2) one or more of sodium hydroxide and potassium hydroxide: calcium, zinc, magnesium, iron, and potassium; b) A step of co-mixing the one or more edible proteins and one or more edible polysaccharides in the one or more solvents to form a mixture; c) a step of extruding the mixture into the forming bath to form extruded hollow fibers, or casting the mixture to form a sheet membrane; d) a step of exposing the extruded hollow fibers or sheet membrane to an energy source selected from one or more heat and irradiation sufficient to at least partially crosslink the one or more proteins to form edible crosslinked porous hollow fibers; A method that includes this.

35. A hollow fiber or sheet membrane prepared by the method described in claim 34.

36. Any of claims 1 to 35, wherein one or more proteins, one or more polysaccharides, one or more solvents, plasticizers and / or one or more components of the forming bath are generally recognized as safe (GRAS) by the U.S. Food and Drug Administration (FDA).

37. The obtained sheet film or hollow fiber undergoes an exchange of 10-50% water for glycerol in order to dry without pore collapse, according to any one of claims 1 to 36.

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